Tyrosine kinase receptors and ligands

ABSTRACT

The present invention provides for a gene, designated as musk, that encodes a novel tyrosine kinase receptor expressed in high levels in denervated muscle. The invention also provides for an isolated polypeptide which activates MuSK receptor. The invention further provides for a polypeptide which is functionally equivalent to the MuSK activating polypeptide. The invention also provides assay systems that may be used to detect and/or measure ligands that bind the musk gene product. The present invention also provides for diagnostic and therapeutic methods based on molecules that activate MuSK.

This application is a divisional application of U.S. Ser. No. 09/077,955filed 10 Sep. 1998, now U.S. Pat. No. 6,413,740, which is the NationalStage of International Application No. PCT/US96/20696, filed 13 Dec.1996, which is a continuation-in-part of U.S. Ser. No. 08/644,271, filed10 May 1996, now U.S. Pat. No. 5,818,478, which claims the benefit ofProvisional application U.S. Ser. No. 60/008,657, filed 15 Dec. 1995,all of which are incorporated herein by reference.

Throughout this application various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application.

INTRODUCTION

The present invention provides for a novel receptor molecule, a novelmolecule capable of activating the receptor, and methods of making anduse thereof.

BACKGROUND OF THE INVENTION

The ability of polypeptide ligands to bind cells and thereby elicit aphenotypic response such as cell growth, survival or differentiation insuch cells is often mediated through receptor tyrosine kinases. Theextracellular portion of each receptor tyrosine kinase (RTK) isgenerally the most distinctive portion of the molecule, as it providesthe protein with its ligand-recognizing characteristic. Binding of aligand to the extracellular domain results in signal transduction via anintracellular tyrosine kinase catalytic domain which transmits abiological signal to intracellular target proteins. The particular arrayof sequence motifs of this cytoplasmic, catalytic domain determines itsaccess to potential kinase substrates (Mohammadi, et al., 1990, Mol.Cell. Biol., 11: 5068-5078; Fantl, et al., 1992, Cell, 69:413-413).

The tissue distribution of a particular tyrosine kinase receptor withinhigher organisms provides relevant data as to the biological function ofthe receptor. For example, the localization of a Trk family receptor,TrkB, in tissue provided some insight into the potential biological roleof this receptor, as well as the ligands that bind this receptor(referred to herein as cognates). Thus, for example, in adult mice, trkBwas found to be preferentially expressed in brain tissue, althoughsignificant levels of trkB mRNAs were also observed in lung, muscle, andovaries. Further, trkB transcripts were detected in mid and lategestation embryos. In situ hybridization analysis of 14 and 18 day oldmouse embryos indicated that trkB transcripts were localized in thecentral and peripheral nervous systems, including brain, spinal cord,spinal and cranial ganglia, paravertebral trunk of the sympatheticnervous system and various innervation pathways, suggesting that thetrkB gene product may be a receptor involved in neurogenesis and earlyneural development as well as play a role in the adult nervous system.

The cellular environment in which an RTK is expressed may influence thebiological response exhibited upon binding of a ligand to the receptor.Thus, for example, when a neuronal cell expressing a Trk receptor isexposed to a neurotrophin which binds that receptor, neuronal survivaland differentiation results. When the same receptor is expressed by afibroblast, exposure to the neurotrophin results in proliferation of thefibroblast (Glass, et al., 1991, Cell 66:405-413). Thus, it appears thatthe extracellular domain provides the determining factor as to theligand specificity, and once signal transduction is initiated thecellular environment will determine the phenotypic outcome of thatsignal transduction.

A number of RTK families have been identified based on sequencehomologies of their intracellular domains. For example, two members ofthe TIE (tyrosine kinase with immunoglobulin and EGF homology domains)family, known as TIE-1 and TIE-2, have 79% sequence homology in theirintracellular region (Maisonpierre, et al., 1993, Oncogene 8:1631-1637).Although these receptors share similar motifs in their extracellulardomain, only 32% of the sequences are identical.

A receptor having a kinase domain that is related to the Trk family hasbeen identified in the electric ray Torpedo californica and may play arole in motor neuron induced synapses on muscle fibers. Jennings, et al.Proc. Natl. Acad. Sci. USA 90: 2895-2899 (1993). This kinase wasisolated from the electric organ, a tissue which is a specialized formof skeletal muscle. The tyrosine kinase domain of this protein isrelated to the Trk family, while the extracellular domain is somewhatdivergent from the Trks. The protein was found to be expressed at highlevels in Torpedo skeletal muscle, and at much lower levels in adultTorpedo brain, spinal cord, heart, liver and testis.

Often such novel RTKs are identified and isolated by searching foradditional members of known families of tyrosine kinase receptors using,for example, PCR-based screens involving known regions of homology amongTrk family members. (See, for example, Maisonpierre, et al., 1993,Oncogene 8: 1631-1637). Isolation of such so called “orphan” tyrosinekinase receptors, for which no ligand is known, and subsequentdetermination of the tissues in which such receptors are expressed,provides insight into the regulation of the growth, proliferation andregeneration of cells in target tissues. The identification andisolation of novel RTKs may be used as a means of identifying newligands or activating molecules that may then be used to regulate thesurvival, growth, differentiation and/or regeneration of cellsexpressing the receptors. Further, because RTKs appear to mediate anumber of important functions during development, the identification andisolation of such receptors, ligands and activating molecules enhancesour understanding of developmental processes and may improve our abilityto diagnose or treat abnormal conditions.

For example, the above described methods may be used to study an eventthat occurs during development of the neuromuscular junction (NMJ)—thelocalization of acetylcholine receptors at the synapse. It has long beenknown that important signals are exchanged across the NMJ (Nitkin etal., 1987, J.Cell.Biol. 105: 2471-2478; Hall, Z. W. and Sanes, J. R.,1993, Cell/Neuron (Suppl.) 72/10: 99-121; Bowe, M. A. and Fallon, J. R.,1995, Ann. Rev. Neurosci. 18: 443-462; Sanes, J. R., 1995, Devel. Biol.6: 163-173; Burden, S. J., et al., 1995, Devel. Biol. 6: 59-65). Thesesignals include the chemical transmitter, acetylcholine, which isreleased from vesicles in the nerve terminal, recognized byacetylcholine receptors (AChRs) on the muscle, and ultimately results inelectrical activation and contraction of the muscle.

Muscle also provides neurotrophic factors that support survival of motorneurons (DeChiara, T. et al., 1995, Cell 83: 313-322), and the nerve mayin turn provide myotrophic factors that maintain muscle mass (Helgren,M. E., et al., 1994, Cell 76: 493-504). Reciprocal signalinginteractions are also critical both for the formation and maintenance ofthe neuromuscular junction itself. Such signals regulate recognition ofnerve-to-muscle contact, arrest the growth of the incoming nerve ending,and induce formation of a highly specialized nerve terminal marked by apolarized arrangement of synaptic vesicles and active zones.Simultaneously, precisely juxtaposed with respect to the nerve terminal,a complex molecular apparatus forms on the muscle membrane. Thisspecialized postsynaptic structure, termed the motor endplate, comprisesa tiny patch on the muscle membrane which is characterized by a denseclustering of particular proteins; some of these may receivenerve-derived signals, as AChRs are known to do, while others may beinvolved in creating the molecular scaffold for this post-synapticspecialization.

Signals produced by the nerve induce postsynaptic clusters by at leasttwo mechanisms. First, these signals can induce redistribution ofpre-existing molecules that are initially expressed throughout themyofiber, and second, they can induce localized transcription ofspecific genes only by subsynaptic nuclei underlying the NMJ. Betweenthe nerve terminal and the motor endplate is a narrow synaptic cleftcontaining a complex basal lamina. This basal lamina is distinguishedfrom the adjacent extracellular matrix by the accumulation of a numberof proteins, such as acetylcholinesterase and s-laminin. The synapticbasal lamina also serves as a reservoir for signaling moleculesexchanged between nerve and muscle.

While the reciprocal interactions between nerve and muscle have beenintensively explored for decades, many questions still remain concerningthe precise nature of the signals involved in formation of the NMJ. Therealization that empty sheaths of the synaptic basal lamina could induceformation of both nerve terminal specializations and motor endplatessuggested that key signaling molecules might be embedded in theextracellular matrix (Sanes, J. R. et al., 1978, J.Cell. Biol. 78:176-198; Burden, S. J., et al., 1979, J.Cell. Biol. 82: 412-425;McMahan, U. J. and Slater, C. R., 1984, J.Cell. Biol. 98: 1453-1473;Kuffler, D. P., 1986, J.Comp. Neurol. 250: 228-235). Indeed, recentfindings indicate that a protein discovered for its AChR-inducingactivity and thus termed ARIA (Jessell, T. M., et al., 1979, PNAS (USA)76: 5397-5401; Falls, D. L., et al., 1990, Cold Spring Harbor Symp.Quant. Biol. 55: 397-406; Falls, D. L., et al., 1993, Cell 72: 801-815)which can increase the expression of several of the AChR subunit genes(Harris, D. A., 1989, et al., Nature 337: 173-176; Martinou, J.-C., etal., 1991, PNAS (USA) 88: 7669-7673; Jo, S. A., et al., 1995, Nature373: 158-161; Chu, G. C., et al., 1995, Neuron 14: 329-339), islocalized to the synaptic basal lamina (Jo, S. A., et al., 1995, Nature373: 158-161; Goodearl, A. D., et al., 1995, J.Cell. Biol. 130:1423-1434). Molecular cloning has revealed that ARIA corresponds to afactor alternatively referred to as neuregulin, NDF, heregulin or gliagrowth factor, and binds to the erbB family of RTKs (Carraway, K. L. andBurden, S. J., 1995, Curr. Opin. Neurobiol. 5: 606-612). Interestingly,neuregulin production has been demonstrated in motor neurons andneuregulin receptors, erbB3 and erbB4, have recently been localized tothe motor endplate, supporting the idea that nerve-derived neuregulinprovides an important signal to muscle that regulates transcription fromsubsynaptic nuclei (Altiok, N., et al., 1995, EMBO J. 14: 4258-4266;Moscoso, L. M., et al., 1995, Dev. Biol. 172: 158-169; Zhu, X., et al.,1995, EMBO J. 14: 5842-5848).

Another protein, known as agrin, was isolated from the synaptic basallamina based on its ability to cause redistribution of pre-existingAChRs into clusters on the surface of cultured myotubes (Godfrey, E. W.,et al., 1984, J.Cell. Biol. 99: 615-627; Rupp, F., et al., 1991, Neuron6: 811-823; Tsim, K. W., et al., 1992, Neuron 8: 677-689). In contrastto neuregulin, agrin does not appear to regulate AChR expression.However, agrin causes the clustering of a number of synaptic components,along with AChRs, in cultured myotubes (Wallace, B. G., 1989,J.Neurosci. 9: 1294-1302).

A variety of data are consistent with the notion that agrin also acts invivo to induce and maintain the postsynaptic membrane specialization.Most important among these are the findings that the most active formsof agrin are exclusively made by neurons and are deposited in thesynaptic basal lamina (Ruegg, M. A., et al., 1992, Neuron 8: 691-699;Ferns, M., et al., 1993, Neuron 11: 491-502; Hoch, W., et al., 1993,Neuron 11: 479-490), and that antibodies to agrin block nerve-inducedclustering of AChRs on cultured myotubes (Reist, N. E., et al., 1992,Neuron 8: 865-868).

The precise mechanism of action of agrin remains a mystery (Sealock, R.and Froehner, S. C., 1994, Cell 77: 617-619). Agrin is known to inducetyrosine phosphorylation of AChRs, and inhibitors of tyrosinephosphorylation block agrin-mediated clustering (Wallace, B. G., et al.,1991, Neuron 6: 869-878; Wallace, B. G., 1994, J.Cell. Biol. 125:661-668; Qu, Z. and Huganir, R. L., 1994, J.Neurosci. 14: 6834-6841;Wallace, B. G., 1995, J.Cell. Biol. 128: 1121-1129).

Intriguing recent findings have revealed that agrin can directly bind toα-dystroglycan, an extrinsic peripheral membrane protein that isattached to the cell surface by covalent linkage to β-dystroglycan,which in turn couples to the intracellular cytoskeletal scaffold via anassociated protein complex (Bowe, M. A., et al, 1994, Neuron 12:1173-1180; Campanelli, J. T., et al., 1994, Cell. 77: 673-674; Gee, S.H., et al., 1994, Cell 77: 675-686; Sugiyama, J., et al., 1994, Neuron13: 103-115; Sealock, R. and Froehner, S. C., 1994, Cell 77: 617-619).

Extrasynaptically, the dystroglycan complex binds laminin on itsextracellular face, and couples to the actin scaffold via aspectrin-like molecule known as dystrophin. At the synapse however,agrin (via its own laminin-like domains) may be able to substitute forlaminin, whereas utrophin (a dystrophin related protein) replacesdystrophin as the link to actin (reviewed in (Bowe, M. A. and Fallon, J.R., 1995, Ann. Rev. Neurosci. 18: 443-462)). The dystroglycan complexco-clusters with AChRs in response to agrin in vitro and components ofthis complex are concentrated at the endplate in vivo (reviewed in(Bowe, M. A. and Fallon, J. R., 1995, Ann. Rev. Neurosci. 18: 443-462)).

Recent evidence suggests that a 43 kD cytoplasmic protein, known asrapsyn, anchors AChRs to a sub-synaptic cytoskeleton complex, probablyvia interactions with the dystroglycan complex (Cartaud, J. andChangeux, J. P., 1993, Eur. J. Neurosci. 5: 191-202; Apel, E. D., etal., 1995, Neuron 15: 115-126). Gene disruption studies reveal thatrapsyn is absolutely necessary for clustering of AChRs, as well as ofthe dystroglycan complex. However, other aspects of NMJ formation,involving presynaptic differentiation and synapse-specifictranscription, are seen in mice lacking rapsyn (Gautam, M., et al.,1995, Nature 377: 232-236).

Despite the findings that agrin can bind directly to α-dystroglycan, andthat AChRs and the dystroglycan complex are linked and co-cluster inresponse to agrin, the role of dystroglycan as an agrin receptor remainsunclear (Sealock, R. and Froehner, S. C., 1994, Cell 77: 617-619; Ferns,M., et al., 1996, J. Cell Biol. 132: 937-944). It has recently beenreported that a 21 kD fragment of chick agrin is sufficient to induceAChR aggregation (Gesemann, M., et al., 1995, J. Cell. Biol. 128:625-636). Dystroglycan could be directly involved in activatingsignaling pathways that appear to be required for clustering, such asthose involving tyrosine phosphorylation, by an unknown mechanism (forexample, via association with a cytoplasmic tyrosine kinase).

Alternatively, dystroglycan could be involved in couplings of agrin notonly to AChRs but to a novel signaling receptor. It also remainspossible that dystroglycan does not play an active or required role ininitiating clustering, and is merely among an assortment ofpost-synaptic molecules that undergo clustering. Recent evidenceindicates that the agrin fragment that is active in inducing AChRaggregation does not bind to α-dystroglycan and a structural role inaggregation, rather than a signal transfer role, has been proposed forthe binding of agrin to α-dystroglycan (Gesemann, M., et al., 1996,Neuron 16: 755-767).

SUMMARY OF THE INVENTION

The present invention provides for a novel tyrosine kinase, termed“MuSK” for “muscle specific kinase,” that is expressed in normal anddenervated muscle, as well as other tissues including heart, spleen,ovary or retina (See Valenzuela, D., et al., 1995, Neuron 15: 573-584).The novel tyrosine kinase has alternatively been referred to as “Dmk”for “denervated muscle kinase.” (See PCT International Application No.PCT/US94/08039 published Feb. 1, 1996 as WO 96/02643 entitled DenervatedMuscle Kinase (DMK), A Receptor Of The Tyrosine Kinase Superfamily).Thus, the terms MuSK and Dmk may be used interchangeably. The proteinappears to be related to the Trk family of tyrosine kinases.

The present invention further provides for an isolated nucleic acidmolecule encoding MuSK.

The present invention also provides for a protein or peptide thatcomprises the extracellular domain of MuSK and the nucleic acid whichencodes such extracellular domain. The invention further provides forvectors comprising an isolated nucleic acid molecule encoding MuSK orits extracellular domain, which can be used to express MuSK in bacteria,yeast and mammalian cells.

The present invention also provides for use of the MuSK receptor or itsextracellular or intracellular domain to screen for drugs that interactwith or activate MuSK. Novel agents that bind to and/or activate thereceptor described herein may mediate survival, proliferation anddifferentiation in cells naturally expressing the receptor, but also maymediate survival, proliferation or differentiation when used to treatcells engineered to express the receptor.

In particular embodiments, the extracellular domain (soluble receptor)of MuSK is utilized in screens for cognate ligands and activatingmolecules. For example, the MuSK receptor activating molecule describedherein may be used in a competition assay to identify agents capable ofacting as receptor agonists or antagonists by competing the agents withMuSK activating molecule for phosphorylation of the MuSK receptor.Specifically, the active portion of human agrin described herein may beused as the MuSK activating molecule in a competition assay to screenfor agents capable of acting as receptor agonists or antagonists.

The term “MuSK activating molecule” as used herein refers to a moleculewhich is capable of inducing phosphorylation of the MuSK receptor in thecontext of a differentiated muscle cell. One such activating molecule isagrin as described in the Examples set forth herein.

The present invention also provides for nucleic acid probes, capable ofhybridizing with a sequence included within the nucleotide sequenceencoding human MuSK or its activating molecule, useful for the detectionof MuSK expressing tissue or MuSK activating molecule-expressing tissuein humans and animals. The invention further provides for antibodiescapable of specifically binding MuSK or MuSK activating molecule. Theantibodies may be polyclonal or monoclonal.

The present invention also has diagnostic and therapeutic utilities. Inparticular embodiments of the invention, methods of detectingaberrancies in the function or expression of the receptor describedherein may be used in the diagnosis of muscular or other disorders. Inother embodiments, manipulation of the receptor, agonists which bindthis receptor, or receptor activating molecules may be used in thetreatment of neurological diseases or diseases of muscle orneuromuscular unit disorders, including, but not limited to, musculardystrophy and muscle atrophy. In further embodiments, the extracellulardomain of the receptor is utilized as a blocking agent.

The present invention also provides for an isolated and purifiedpolypeptide which activates MuSK receptor. In one embodiment, thepolypeptide of the invention is encoded by a nucleotide sequencecomprising the coding region of the active portion of human agrincontained in the vector designated as pBluescript human Agrin-1(pBL-hAgrin1) that was deposited with the American Type CultureCollection on Dec. 12, 1995 under ATCC Accession No. 97378. The presentinvention further provides for an isolated polypeptide which isfunctionally equivalent to this polypeptide.

The invention further provides for an isolated and purified nucleic acidmolecule comprising a nucleotide sequence encoding the active portion ofhuman agrin, wherein the nucleotide sequence is selected from the groupconsisting of:

-   (a) the nucleotide sequence comprising the coding region of the    active portion of human agrin contained in the vector designated as    pBL-hAgrin 1 (ATCC Accession No. 97378);-   (b) a nucleotide sequence that hybridizes under stringent conditions    to the nucleotide sequence of (a) and which encodes the active    portion of human agrin; and-   (c) a nucleotide sequence that, as a result of the degeneracy of the    genetic code, differs from the nucleotide sequence of (a) or (b) and    which encodes the active portion of human agrin.

The invention also provides for the above-described nucleic acidmolecule which additionally contains a nucleotide sequence so that theencoded polypeptide contains the eight amino acids ELANEIPV at theposition corresponding to amino acid position 1780 as shown in FIGS.14A-14C (SEQ ID NO: 34).

The invention also provides for a method of promoting the growth,survival or differentiation of a MuSK receptor expressing cellcomprising administering to the MuSK receptor expressing cell aneffective amount of agrin or a derivative of agrin. The method may bepracticed in vitro or in vivo. In one embodiment of this method, theagrin is human agrin. In another embodiment of this method, the MuSKreceptor expressing cell is a cell which is normally found in the heart,spleen, ovary, retina or skeletal muscle. In another embodiment, theMuSK receptor expressing cell is a cell which has been geneticallyengineered to express the MuSK receptor.

The present invention also includes a method of treating a patientsuffering from a muscle disease or neuromuscular disorder comprisingadministering to the patient an effective amount of agrin or aderivative thereof. By way of non-limiting example, the agrin may behuman agrin and the derivative may be the active portion of the humanagrin molecule.

The present invention also includes an antibody capable of specificallybinding human agrin. More specifically, the invention includes anantibody capable of specifically binding the active portion of humanagrin. The antibody may be monoclonal or polyclonal. The inventionfurther provides a method of detecting the presence of human agrin in asample comprising:

-   -   a) reacting the sample with an antibody capable of specifically        binding human agrin under conditions whereby the antibody binds        to human agrin present in the sample; and    -   b) detecting the bound antibody, thereby detecting the presence        of human agrin in the sample.

The antibody used may be monoclonal or polyclonal. The sample may bebiological tissue or body fluid. The biological tissue may be brain,muscle, or spinal cord. The body fluid may be cerebrospinal fluid,urine, saliva, blood, or a blood fraction such as serum or plasma.

The invention further provides for an isolated and purified nucleic acidmolecule comprising a nucleotide sequence encoding human muscle specifickinase (MuSK) receptor, wherein the nucleotide sequence is selected fromthe group consisting of:

-   (a) the nucleotide sequence comprising the coding region of the    human MuSK receptor as set forth in FIGS. 4A-4D (SEQ ID NO: 32);-   (b) a nucleotide sequence that hybridizes under stringent conditions    to the nucleotide sequence of (a) and which encodes a human MuSK    receptor; and-   (c) a nucleotide sequence that, as a result of the degeneracy of the    genetic code, differs from the nucleotide sequence of (a) or (b) and    which encodes a human MUSK receptor.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1D—Nucleotide (SEQ ID NO: 2) and deduced amino acid (singleletter code) sequences (SEQ ID NO: 1) of rat musk. The nucleotidesequence encoding mature MuSK begins around nucleotide 192.

FIG. 2—Northern blot showing distribution of musk in the rat duringearly development. Lane 1: Total embryo E9; Lane 2: Total embryo E11;Lane 3: Placenta E11; Lane 4: Embryo head E12; Lane 5: Embryo body E12;Lane 6: Embryo spinal cord E12; Lane 7: Placenta E12; Lane 8: Embryohead E13; Lane 9: Embryo body E13; Lane 10: Embryo brain E17; Lane 11:Embryo brain P1; Lane 12: Embryo brain P10; Lane 13: Embryo brain P19;Lane 14: Adult brain; Lane 15: Adult muscle; Lane 16: Adult denervatedmuscle; where day of sperm positivity is designated as day E1, and dayof birth is designated as day P1.

FIG. 3—Northern blot showing distribution of musk in adult rat tissues.Lane 1: Brain; Lane 2: Olfactory bulb; Lane 3: Cortex; Lane 4:Hippocampus; Lane 5: Thalamus/hypothalamus; Lane 6: Midbrain; Lane 7:Hindbrain; Lane 8: Cerebellum; Lane 9: Spinal Cord; Lane 10: Thymus;Lane 11: Spleen; Lane 12: Liver; Lane 13: Kidney; Lane 14: Lung; Lane15: Sciatic Nerve; Lane 16: Retina; Lane 17: Heart; Lane 18: Ovary; Lane19: Muscle; Lane 20: Denervated muscle.

FIGS. 4A-4D—Nucleotide (SEQ ID NO: 32) and deduced amino acid (singleletter code) sequences (SEQ ID NO: 33) of human MuSK receptor.

FIG. 5—Schematic representation of genomic DNA encompassing the threekinase domain exons of the mouse MuSK gene, of the targeting vectorconstructed, and of a mutant locus following successful targeting. Thethree exons of the MuSK kinase domain are indicated as black boxes,containing the indicated kinase subdomains (SD). The PGK-neo and MC1-tkcassettes are indicated as open boxes. The novel EcoRI (R) and NcoI (N)fragments generated following successful targeting are labeled. The 5′EcoRI/HpaI probe used to detect the endogenous and mutant EcoRIfragments was derived from genomic DNA not included in the targetingconstruct. B, BamHI; Hp, HpaI; S, SpeI (sites included withinparentheses are destroyed in the cloning process).

FIG. 6—MuSK Knockout Mice—Southern blot of tail DNA from wild-type,heterozygous and homozygous F2 progeny showing the endogenous and mutantEcoRI fragments detected by the 5′ RI/HpaI probe, as well as theendogenous NcoI fragments detected by the kinase region probe, which areabsent in the homozygous mutant.

FIGS. 7A-7D—Post-mortem histological analysis of lung demonstrating thatthe alveoli air sacs in the MuSK^(−/−) newborn are not expanded (FIG.7A) as they are in the lung of the control littermate (FIG. 7B),indicating that mutant pups do not take a single breath. Post-mortemhistological analysis of hindlimb musculature reveals that MuSK^(−/−)mice (FIG. 7C) possess grossly normal muscle architecture similar tothat of control mice (FIG. 7D).

FIGS. 8A-8C—Agrin induces AChR clustering in myotubes from control butnot MuSK^(−/−) mice. Myotubes derived from control and MuSK^(−/−) micewere treated overnight with varying concentrations of agrin_(4,8),stained with rhodamine-conjugated α-bungarotoxin (α-BGT) to labelsurface AChRs, and then either photographed at 64× magnification underrhodamine optics (FIG. 8A, challenge with 100 nM agrin depicted) orsubjected to AChR cluster quantitation (FIG. 8B, each point representsthe mean±SEM of forty myotube segments). Total AChRs on the myotubesbefore agrin treatment was determined by binding with ¹²⁵I-α-BGT (FIG.8C, each bar represents the mean±SEM CPM bound per μg of total cellprotein (control: N=6; MuSK−/−: N=5).

FIGS. 9A-9D—c-agrin_(4,8) and c-agrin_(0,8) specifically induce rapidtyrosine phosphorylation of MuSK receptors. C2C12 and primary ratmyoblasts were differentiated into myotubes and stimulated withconditioned media from COS cells transfected with a plasmid control(Mock) or plasmids encoding the various forms of soluble agrin, withconditioned media containing neuregulin, or with purified bFGF orinsulin, as labelled. Stimulations were for ten minutes using 10 nMconcentrations of the various factors, except as indicated in FIGS. 9Cand 9D. Following factor challenges, the cells were lysed and subjectedto immunoprecipitations (I.P.) for either the MuSK or ErbB3 receptors asindicated, then immunoblotted for phosphotyrosine levels. Only agrinscontaining the eight amino acid insert at the Z position, but not otherfactors, could induce MuSK phosphorylation (FIG. 9A). Agrin could notinduce phosphorylation of another muscle receptor, ErbB3 (FIG. 9B). MuSKphosphorylation occurred at low agrin concentrations (FIG. 9C) and veryrapidly in response to agrin (FIG. 9D).

FIGS. 10A & 10B—Agrin can not detectably bind to the isolated ectodomainof MuSK. Agrin was assayed for its binding to immobilized MuSK-Fc or toan immoblized agrin-specific monoclonal antibody (mAb), each coupled toa BIAcore sensorchip surface (FIG. 10A); bindings to the MuSK-Fc surfacewere also done in the presence 2 mM Ca⁺⁺ or heparin (0.01 mg/ml), asindicated, while bindings to the antibody surface were also competedwith excess soluble monoclonal antibody or MuSK-Fc (each at 25 μg/ml),as indicated. Reciprocally, binding of soluble MuSK-Fc or monoclonalantibody to immobilized agrin was assayed by first binding conditionedmedia transfected with a plasmid control (Mock) or a plasmid encodingc-agrin4,8 (cAg_(4,8)) to nitrocellulose, followed by detection usingeither the soluble MuSK-Fc or the agrin-specific monoclonal antibody, asindicated (FIG. 10B); TrkB-Fc detection of nitrocellulose-immobilizedBDNF served as an additional control.

FIG. 11—Agrin can only induce MuSK phosphorylation in the context of adifferentiated myotube: evidence for a myotube-specific accessorycomponent. Agrin-inducible phosphorylation of an introduced chick MuSKreceptor was evaluated in a clone of C2C12 myoblasts stably transfectedwith a chick MuSK expression vector. The introduced chick MuSK isexpressed regardless of whether this C2C12 clone is undifferentiated(“Undif”) or differentiated into myotubes (“Dif”) (middle panel), incontrast to the endogenous mouse MuSK, which is only expressed indifferentiated cells (bottom panel). However, the chick MuSK can only beinducibly phosphorylated in response to agrin when it is assayed indifferentiated myotubes (top panel). The chick MuSK displays the samespecificity for activation by the various agrin isoforms (each at 10 nMfor ten minutes) as does the endogenous mouse MuSK (compare transfectedchick MuSK and endogenous mouse MuSK in upper panel).

FIGS. 12A-12C. Relevant models for the agrin/MuSK receptor complex. FIG.12A—Schematic representation depicting the step-wise assembly of themulti-component receptor complex for ciliary neurotrophic factor (CNTF);b1, gp130; b2, LIFRb. FIG. 12B—Schematic depiction of the use of solubleb receptor components (Fc-tagged) to build a CNTF receptor complexattached to the cell surface via only one of its components, thenon-signaling a component; surface binding of the soluble b componentscan be detected using antibodies recognizing the Fc tag. FIG.12C—Schematic representation of one of several possible models of theMuSK receptor complex for agrin, depicting requirement for amyotube-associated specificity component (M.A.S.C.) and possibleinteractions to additional components that may be required for signalingor coupling to various effectors or substrates; these couplings may bemediated extracellularly (for example via agrin binding to thedystroglycan complex) or intracellularly (for example via interactionsof SH2 domain-containing proteins to phosphorylated tyrosines on MuSK).

FIGS. 13A-13C. Evidence for an agrin/MuSK receptor complex utilizing amyotube-specific accessory component. FIG. 13A—Formation of agrin/MuSKcomplexes on the surface of myotubes: undifferentiated (Undiff.) ormyotube-differentiated (Diff) C2C12 cells were assayed for their abilityto bind either MuSK-Fc or a control receptor-Fc fusion (TrkB-Fc), in theabsence or presence of various agrin isoforms (provided in conditionedmedia from transient COS transfections); specific binding of MuSK-Fc tothe myotube surface, which is enhanced by exogenously provided agrin, issuggested to involve complexes analogous to those depicted in FIG. 12B.FIG. 13B—Direct binding of agrin to MuSK is demonstrated bycross-linking analysis. Radiolabelled agrin (a recombinant c-terminalfragment (or portion) of human agrin, termed hAgrin_(4,8)) at 1 nM waschemically cross-linked to the surface of myotubes. Followingcross-linking, lysates were immunoprecipitated with a MuSK-specificantibody (lane 1). The cross-linking was also done in the presence ofexcess (150 nM) unlabelled agrin (lane 2), while the immunoprecipitationwas also done in the presence of excess peptide (corresponding to thatused to generate the antibody) to block the MuSK precipitation;positions of the agrin/MuSK complex, as well as of various forms ofunbound monomeric and dimeric agrin (see text), are indicated. FIG.13C—Inhibition of agrin-induced AChR clustering by MuSK-Fc:agrin-induced AChR-clustering (using 10 nM c-agrin_(4,8)) was performedon C2C12 myotube cultures in the presence of varying concentrations ofsoluble MuSK-Fc or a control receptor-Fc fusion (Ret-Fc); the solubleMuSK-Fc specifically inhibits, presumably by forming inactive complexeson the cell surface with agrin and the myotube-specific accessorycomponent.

FIGS. 14A-14C—Amino acid (single letter code) sequence (SEQ ID NO: 34)of rat agrin indicating Y and Z sites of amino acid inserts found insplice variants.

FIGS. 15A-15B—Nucleotide (SEQ ID NO: 35) and amino acid (single lettercode) sequences (SEQ ID NO: 36) of human agrin expression constructincluding the signal peptide and flg tag (FLAG tag). The start of thecoding region for the active C-terminal fragment (portion) of humanagrin 4-8 is indicated. Also indicated are the position Y and position Zinsert sites at which the 4 and 8 amino acid inserts are located.Throughout this application, references to human agrin 4,8; c-agrin 4,8;or human c-agrin 4,8 indicate the active C-terminal fragment (portion)of human agrin 4-8 as set forth in the Figure.

FIG. 16—Results of phosphorylation assay showing that the activeC-terminal 50 kD portion of human agrin 4,8 and the truncated delta 9portion of human agrin can each induce phosphorylation of the MuSKreceptor.

FIG. 17—Results of pharmacokinetic study comparing serum half-lives ofactive C-terminal 50 kD portion of human agrin 4,8 (c-agrin 4,8) withactive C-terminal 50 kD portion of human agrin 4,8 that has beenmodified by covalent addition of polyethylene glycol.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides for a novel tyrosine kinase molecule thatis related to the trk family of tyrosine kinases. The sequence of theprotein is set forth in FIGS. 1A-1D as SEQ. ID NO: 1. The coding regionof the mature protein is believed to begin on or around theserine-glycine-threonine on or around position 20 of the coded region.

The novel tyrosine kinase described herein has been found to be inducedin denervated skeletal muscle. Accordingly, it has been designated asMuSK (muscle specific kinase). It has also been referred to previouslyas Dmk (denervated muscle kinase). In addition to being found inskeletal muscle, both normal and denervated, MuSK has also been found tobe present in, but not be limited to, the spleen, ovary and retina. Itappears to be present during early development, but is also found inadult tissue.

MuSK may be related to the Torpedo RTK identified by Jennings, et al.supra. However, MuSK differs in that it appears to be induced indenervated muscle, whereas no such induction has been reported withregard to the Torpedo RTK. Furthermore, the Torpedo RTK has anextracellular kringle domain, whereas MuSK does not. However, thesekinases may be members of the same or related families.

The gene encoding rat MuSK has been cloned and the DNA sequencedetermined (FIGS. 1A-1D; SEQ ID NO: 2). The extracellular domain of themature protein is believed to be encoded by the nucleotide sequencebeginning on or around position 192 and ending on or arQund position1610. The transmembrane portion of the protein is believed to be encodedby the nucleotide sequence beginning on or around position 1611 andending on or around position 1697. The intracellular domain is believedto be encoded by the nucleotide sequence beginning on or around position1698 and ending on or around position 2738. A cDNA clone encoding Dmk(MuSK) was deposited with the American Type Culture Collection on Jul.13, 1993 and accorded an accession number of ATCC No. 75498.

The present invention also provides for a protein or peptide thatcomprises the extracellular domain of MuSK as well as the sequence ofnucleotides which encode this extracellular domain. The extracellulardomain of the protein is believed to be comprised of the amino acids ator around positions 20 through 492 of the coding region set forth as SEQID NO: 1.

The similarity between MuSK and the Torpedo RTK suggests the utilizationof regions of sequence homologies within these genes to develop primersuseful for searching for additional, related RTKs.

Accordingly, the invention provides for nucleic acids, oroligonucleotides greater than about 10 bases in length, that hybridizeto the nucleic acid sequences described herein and that remain stablybound under stringent conditions. Stringent conditions as used hereinare those which (1) employ low ionic strength and high temperature forwashing, for example, 0.15 M NaCl/0.015 M sodium citrate/0.1% NaDodSO₄at 50° C., or (2) use during hybridization of a denaturing agent such asformamide, for example, 50% (vol/vol) formamide with 0.1% bovine serumalbumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphatebuffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate at 42° C.

The present invention further provides for an isolated and purifiednucleic acid molecule comprising a nucleotide sequence encoding humanmuscle specific kinase (MuSK) receptor, wherein the nucleotide sequenceis selected from the group consisting of:

-   -   (a) the nucleotide sequence comprising the coding region of the        human MuSK receptor as set forth in FIGS. 4A-4D (SEQ ID NO: 32);    -   (b) a nucleotide sequence that hybridizes under stringent        conditions to the nucleotide sequence of (a) and which encodes a        human MuSK receptor; and    -   (c) a nucleotide sequence that, as a result of the degeneracy of        the genetic code, differs from the nucleotide sequence of (a)        or (b) and which encodes a human MuSK receptor.

The invention further provides for isolated and purified human MuSKreceptor encoded by the coding region of the human MuSK receptornucleotide sequence as set forth above. The invention also provides fora vector which comprises the isolated nucleic acid molecule described.In one embodiment, the vector is an expression vector wherein the DNAmolecule is operatively linked to an expression control sequence. In afurther embodiment, the expression vector comprises an immediate earlygene promoter. In a still further embodiment, the expression vector ofthe invention comprises the fos promoter or the jun promoter as theearly gene promoter.

The invention further contemplates a host-vector system for theproduction of a polypeptide having the biological activity of a humanMuSK receptor which comprises the vector described above in a suitablehost cell. By way of nonlimiting example, a suitable host cell may be aC2C12 cell or an NIH3T3 cell. The invention further provides for amethod of producing a polypeptide having the biological activity ofhuman MuSK receptor which comprises growing cells of the above-describedhost-vector system under conditions permitting production of thepolypeptide and recovering the polypeptide so produced.

In addition, the invention provides for a therapeutic compositioncomprising the MuSK receptor activating molecule in a pharmaceuticallyacceptable vehicle.

The invention also provides for an antibody which specifically binds theabove-described MuSK receptor. The antibody of the invention may be apolyclonal or monoclonal antibody.

The invention further provides for a MuSK receptorbody comprising theextracellular portion of the above-described MuSK receptor, fused to animmunoglobulin constant region. In a preferred embodiment, the constantregion of the receptorbody is the human immunoglobulin gamma-1 constantregion (MuSK-IgG1 receptorbody).

The invention further provides a method of detecting the presence ofMuSK ligand in a sample comprising:

-   -   a) reacting the sample with a MuSK receptorbody capable of        specifically binding MuSK ligand under conditions whereby the        MuSK receptorbody binds to MuSK ligand present in the sample;        and    -   b) detecting the bound MuSK receptorbody, thereby detecting the        presence of MuSK ligand in the sample.

The MuSK receptorbody used is most preferably MuSK-IgG1 receptorbody.The sample may be biological tissue or body fluid. The biological tissuemay be muscle, heart, spleen or ovary. The body fluid may becerebrospinal fluid, urine, saliva, blood, or a blood fraction such asserum or plasma.

The invention also provides for a fibroblast cell line that is growthfactor dependent in serum-free medium and that comprises a nucleic acidmolecule encoding the human MuSK receptor as described above.

When using nucleotide sequences coding for part or all of MuSK inaccordance with this invention to isolate new family members or MuSKfrom other species, the length of the sequence should be at leastsufficient to be capable of hybridizing with endogenous mRNA from thevertebrate's own musk. Typically, sufficient sequence size will be about15 consecutive bases (DNA or RNA).

Strategies for identifying novel RTKs using degenerateoligodeoxyribonucleotide primers corresponding to protein regionssurrounding amino acids conserved in tyrosine kinases have beenpreviously described (Wilks, et al., 1989, Proc. Natl. Acad. Sci.U.S.A., 86:1603-1607, Partanen, J. et al., 1990, Proc. Natl. Acad. Sci.U.S.A. 87: 8913-8917; Lai and Lemke, 1991, Neuron 6: 691-704;Masiakowski and Carroll, 1992, J. Biol. Chem. 267: 26181-26190). Thediscovery by applicants of the relationship between MuSK and the TorpedoRTK has led to the identification of heretofore unknown homology regionswhich may be used in screening strategies.

The following primer, based on the amino acid homology domainAsp-Val-Trp-Ala-Tyr-Gly (SEQ ID NO: 3) between MuSK and the Torpedo RTK,may be used in combination with additional primers that correspond toknown homology regions characteristic of RTKs, to isolate relatedtyrosine kinases, e.g. other family members [all codes used hereinrepresenting amino acids and nucleotides are as set forth in 37 C.F.R.§1.822(b)]:

-   5′-GAATTCGAGCTCCCRWANGCCCANACRTC-3′ (SEQ ID NO:4)

The additional primers that correspond to known homology regionscharacteristic of RTKs include the following:

5′ 1) Asp-Leu-Ala-Thr-Arg-Asn     (SEQ ID NO: 5)   5′-TCTTGACTCGAGAYYTNGCNACNMGNAA-3′    (SEQ ID NO: 6) 2)Asp-Leu-Ala-Ala-Arg-Asn    (SEQ ID NO: 7)   5′-TCTTGACTCGAGAYYTNGCNGCNMGNAA-3′    (SEQ ID NO: 8) 3′ 1)Asp-Val-Trp-Ser-Leu-Gly    (SEQ ID NO: 9)   3′-CTRCANACCWSNATRCCCTCGAGCTTAAG-5′    (SEQ ID NO: 10) 2)Asp-Val-Trp-Ser-Phe-Gly    (SEQ ID NO: 11)   3′-CTRCANACCWSNAARCCCTCGAGCTTAAG-5′    (SEQ ID NO: 12) 3)Asp-Val-Trp-Ser-Tyr-Gly    (SEQ ID NO: 13)   3′-CTRCANACCWSNRANCCCTCGAGCTTAAG-5′    (SEQ ID NO: 14)

Alternatively, regions of homology shared by MuSK and members of relatedfamilies, such as the Trk family, may be used in strategies designed toisolate novel RTKs.

The present invention further provides for substantially purifiedprotein molecules comprising the amino acid sequence substantially asset forth in FIGS. 1A-1D for MuSK (SEQ ID NO: 1) or functionallyequivalent molecules. Functionally equivalent molecules includederivatives in which amino acid residues are substituted for residueswithin the sequence resulting in a silent change. For example, one ormore amino acid residues within the sequence can be substituted byanother amino acid of a similar polarity which acts as a functionalequivalent, resulting in a silent alteration. Substitutes for an aminoacid within the sequence may be selected from other members of the classto which the amino acid belongs. For example, the nonpolar (hydrophobic)amino acids include alanine, leucine, isoleucine, valine, proline,phenylalanine, tryptophan and methionine. The polar neutral amino acidsindude glycine, serine, threonine, cysteine, tyrosine, asparagine, andglutamine. The positively charged (basic) amino acids indude arginine,lysine and histidine. The negatively charged (acidic) amino acidsinclude aspartic acid and glutaniic acid. Also included within the scopeof the invention are proteins or fragments (portions) or derivativesthereof which are differentially modified during or after translation,e.g., by glycosylation, proteolytic cleavage, linkage to an antibodymolecule or other cellular ligand, etc.

The invention further contemplates the isolation of proteins that havesubstantial similarity to the MuSK protein described herein. Substantialsimilarity, as used herein, refers to proteins that are from differentspecies or are family members within a species and are identical in atleast 40% of positions. Substantial similarity at the protein levelincludes the ability of a subject protein to compete with MuSK forbinding to monoclonal antibodies raised against MuSK epitopes.

The MuSK protein described herein is useful in 1) screening strategies,2) purification strategies and 3) diagnostic uses. With respect toscreening strategies, expression cloning strategies based on cellsurvival and proliferation assays provide a method of screening forcognate ligands (Glass, et al. (1991) Cell 66:405-413). Since ligandsthat bind MuSK may be membrane bound, other strategies foridentification of such receptors may be more well suited (Armitage, etal. 1992, Nature 357:80-82; Smith, et al. 1993, Cell 73:1349-1360). Inpreferred embodiments, the extracellular domain of MuSK is fused to amarker to create a chimeric protein which enables identification andpurification of the extracellular domain when bound to a cognate.

If, for example, the cognate ligand is membrane bound, as described inSmith, et al. supra, the extracellular portion of MuSK may be fused totruncated immunoglobulin heavy chains (Fc). The fusion product may thenbe used to identify cells expressing surface ligand that binds thereceptor by, for example, flow cytometry. Alternatively, other tags,such as myc used to tag the extracellular domain of MuSK, may also beuseful for the screening and purification of MuSK-binding ligands(Davis, et al. 1991, Science 253:59-63; Squinto, et al., 1990, Neuron5:757-766).

In other embodiments, the extracellular portion of RTKs that bind knownligands are replaced with the extracellular portion of MuSK. Measurableeffects, such as changes in phenotype or induction of early responsegenes, normally associated with binding of the known ligand to thereceptor, can be used to screen for cognate ligands that inducecomparable effects.

For example, a cell line bearing the introduced MuSK receptor or achimeric protein comprising the extracellular domain of MuSK fused tothe transmembrane domain and intracellular domain of another RTK(MuSK-chimeric receptor), as well as the parental cell line without thereceptor can be exposed to any potential source of an agent that mightwork through the receptor. Any specific effects (e.g. on cell survivalor proliferation) on the cell line bearing the receptor or chimera canbe used to identify and eventually purify agents acting on thatreceptor. Once a particular receptor/ligand system is defined, a varietyof additional specific assay systems can be utilized, for example, tosearch for additional agonists or antagonists of MuSK.

According to the invention, MuSK or a MuSK-RTK chimeric receptor, whenintroduced into cells that do not normally express this receptor, can beused to identify ligands that bind the receptor based on thedistinguishable response of the cell. The present invention contemplatesthat the type of response elicited depends on the cell utilized, and notthe specific receptor introduced into the cell. Thus, for example,expression of the MuSK receptor in PC12 pheochromocytoma cells mayresult in the differentiation of the PC12 cells upon exposure to aligand that binds the receptor, whereas the same receptor in fibroblastsmay mediate both survival and proliferation in response to a MuSKbinding ligand. Appropriate cell lines can be chosen to yield a responseof the greatest utility for the assay, as well as discovery of agentsthat can act on tyrosine kinase receptors. “Agents” refers to anymolecule(s), including but not limited to peptide and non-peptidemolecules, that will act in systems to be described in a receptordependent manner.

One of the more useful systems to be exploited involves the introductionof the desired receptor into a growth factor dependent fibroblast cellline. Such a receptor which does not normally mediate proliferativeresponses may, following introduction into fibroblasts, nonetheless beassayed by a variety of well established methods used to quantitateeffects of fibroblast growth factors (e.g. thymidine incorporation orother types of proliferation assays; see van Zoelen, 1990, “The Use ofBiological Assays For Detection Of Polypeptide Growth Factors” inProgress in Factor Research, Vol. 2, pp. 131-152; Zhan and M. Goldfarb,1986, Mol. Cell. Biol., Vol. 6, pp. 3541-3544). These assays have theadded advantage that any preparation can be assayed both on the cellline having the introduced receptor as well as the parental cell linelacking the receptor. Only specific effects on the cell line with thereceptor would be judged as being mediated through the introducedreceptor.

A cell that expresses a receptor described herein may either naturallyexpress the receptor or be genetically engineered to do so. For example,nucleotide sequences obtained as described herein may be introduced intoa cell by transfection, transduction, microinjection, electroporation,via a transgenic animal, etc., using any method known in the art.

The specific binding of test agent to the receptor may be measured in anumber of ways. For example, the binding of test agent to cells may bedetected or measured, by detecting or measuring (i) test agent bound tothe surface of intact cells; (ii) test agent cross-linked to receptorprotein in cell lysates; or (iii) test agent bound to receptor in vitro.The specific interaction between test agent and the receptor may beevaluated by using reagents that demonstrate the unique properties ofthat interaction.

Alternatively, the specific activity of test agent on the receptor maybe measured by evaluating the secondary biological effects of thatactivity, including, but not limited to, the induction of neuritesprouting, immediate early gene expression or phosphorylation of thereceptor. For example, the ability of the test agent to induce neuritesprouting can be tested in cells that lack the receptor and incomparable cells that express, for example, a chimeric receptorcomprising the MuSK extracellular domain and the intracellular domain ofa member of the Trk family (such as TrkA, TrkB or TrkC); neuritesprouting in receptor-expressing cells but not in comparable cells thatlack the receptor would be indicative of a specific test agent/receptorinteraction. A similar analysis could be performed by detectingimmediate early gene (e.g. fos and jun) induction in receptor-minus andreceptor-plus cells, or by detecting phosphorylation of the receptorprotein using standard phosphorylation assays known in the art.

Similarly, the present invention provides for a method of identifying anagent that has signal transducing activity comprising (i) exposing acell that expresses a tyrosine kinase receptor as described herein to atest agent and (ii) detecting the activity of the test agent to thereceptor, in which activity positively correlates with signaltransducing activity. Activity may be detected by either assaying fordirect binding or the secondary biological effects of binding, asdiscussed supra. Such a method may be particularly useful in identifyingnew neurotrophic factors or factors having other pharmaceutical activitysuch as cardioprotective activity, or may be useful in screening a largearray of peptide and non-peptide agents (e.g., peptidomimetics) for suchactivities.

In a preferred, specific, nonlimiting embodiment of the invention, alarge grid of culture wells may be prepared that contain, in alternaterows, PC12 (or fibroblasts, see infra) cells that are eitherreceptor-minus or engineered to be receptor-plus. A variety of testagents may then be added such that each column of the grid, or a portionthereof, contains a different test agent. Each well could then be scoredfor the presence or absence of neurite sprouting. An extremely largenumber of test agents could be screened for signal transducing activityin this manner.

The present invention also provides for assay systems that may be usedaccording to the methods described supra. Such assay systems maycomprise in vitro preparations of receptor, e.g. affixed to a solidsupport, or may preferably comprise cells that express receptor proteinsdescribed herein.

The present invention further provides for host cells and microorganismsand vectors that carry the recombinant nucleic acid molecules describedsupra. Cells that express receptor protein may be genetically engineeredto produce receptor as described supra, by transfection, transduction,electroporation, or microinjection of nucleic acid encoding MuSK in asuitable expression vector. In one embodiment, the host cell carryingthe recombinant nucleic acid is an animal cell, such as COS. In anotherembodiment, the host cell is a bacterium, preferably Escherichia coli.

Any of the methods known to one skilled in the art for the insertion ofDNA fragments into a vector may be used to construct expression vectorsencoding receptor. These methods may include in vitro recombinant DNAand synthetic techniques and in vivo recombinations (geneticrecombination). Expression of nucleotide sequence encoding the receptorprotein or peptide fragment may be regulated by a second nucleotidesequence so that the receptor protein or peptide is expressed in a hosttransformed with the recombinant DNA molecule. For example, expressionof receptor may be controlled by any promoter/enhancer element known inthe art. Promoters which can be used to control receptor expressioninclude, but are not limited to the long terminal repeat as described inSquinto et al., (1991, Cell 65:1-20);

the SV40 early promoter region (Bernoist and Chambon, 1981, Nature290:304-310), the CMV promoter, the M-MuLV 5′ terminal repeat thepromoter contained in the 3′ long terminal repeat of Rous sarcoma virus(Yamamoto, et al., 1980, Cell 22:787-797), the herpes thymidine kinasepromoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. U.S.A.78:144-1445), the regulatory sequences of the metallothioein gene(Brinster et al., 1982, Nature 296:39-42); prokaryotic expressionvectors such as the β-lactamase promoter (Villa-Kamaroff, et al., 1978,Proc. Natl. Acad. Sci. U.S.A. 75:3727-3731), or the tac promoter(DeBoer, et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:21-25). See also“Useful proteins from recombinant bacteria” in Scientific American,1980, 242:74-94; promoter elements from yeast or other fungi such as theGal 4 promoter, the ADH (alcohol dehydrogenase) promoter, PGK(phosphoglycerol kinase) promoter, alkaline phosphatase promoter, andthe following animal transcriptional control regions, which exhibittissue specificity and have been utilized in transgenic animals:elastase I gene control region which is active in pancreatic acinarcells (Swift et al., 1984, Cell 38:639-646; Ornitz et al., 1986, ColdSpring Harbor Symp. Quant. Biol. 50:399-409; MacDonald, 1987, Hepatology7:425-515); insulin gene control region which is active in pancreaticbeta cells (Hanahan, 1985, Nature 315:115-122), immunoglobulin genecontrol region which is active in lymphoid cells (Grosschedl et al.,1984, Cell 38:647-658; Adames et al., 1985, Nature 318:533-538;Alexander et al., 1987, Mol. Cell. Biol. 7:1436-1444), mouse mammarytumor virus control region which is active in testicular, breast,lymphoid and mast cells (Leder et al., 1986, Cell 45:485-495), albumingene control region which is active in liver (Pinkert et al., 1987,Genes and Devel. 1:268-276), alpha-fetoprotein gene control region whichis active in liver (Krumlauf et al., 1985, Mol. Cell. Biol. 5:1639-1648;Hammer et al., 1987, Science 235:53-58); alpha 1-antitrypsin genecontrol region which is active in the liver (Kelsey et al, 1987, Genesand Devel. 1:161-171), beta-globin gene control region which is activein myeloid cells (Mogram et al., 1985, Nature 315:338-340; Kollias etal., 1986, Cell 46:89-94); myelin basic protein gene control regionwhich is active in oligodendrocyte cells in the brain (Readhead et al.,1987, Cell 48:703-712); myosin light chain-2 gene control region whichis active in skeletal muscle (Sani, 1985, Nature 314:283-286), andgonadotropic releasing hormone gene control region which is active inthe hypothalamus (Mason et al., 1986, Science 234:1372-1378).

Expression vectors containing receptor-encoding gene inserts can beidentified by three general approaches: (a) DNA-DNA hybridization, (b)presence or absence of “marker” gene functions, and (c) expression ofinserted sequences. In the first approach, the presence of a foreigngene inserted in an expression vector can be detected by DNA-DNAhybridization using probes comprising sequences that are homologous toan inserted gene. In the second approach, the recombinant vector/hostsystem can be identified and selected based upon the presence or absenceof certain “marker” gene functions (e.g., thymidine kinase activity,resistance to antibiotics, transformation phenotype, occlusion bodyformation in baculovirus, etc.) caused by the insertion of foreign genesin the vector. For example, if the receptor-encoding gene is insertedwithin the marker gene sequence of the vector, recombinants containingthe gene insert can be identified by the absence of the marker genefunction. In the third approach, recombinant expression vectors can beidentified by assaying the foreign gene product expressed by therecombinant vector. Such assays can be based, for example, on thephysical or functional properties of the receptor-encoding gene product,for example, by binding of the receptor to neurotrophic factor or to anantibody which directly recognizes the receptor. Cells of the presentinvention may transiently or, preferably, constitutively and permanentlyexpress receptors or portions thereof.

In preferred embodiments, the present invention provides for cells thatexpress receptors described herein or portions thereof and that alsocontain recombinant nucleic acid comprising an immediate early genepromoter [e.g. the fos or jun promoters (Gilman et al., 1986, Mol. Cell.Biol. 6:4305-4316)]. When such a cell is exposed to a ligand that bindsto the receptor, the binding secondarily induces transcription off theimmediate early promoter. Such a cell may be used to detectreceptor/ligand binding by measuring the transcriptional activity of theimmediate early gene promoter, for example, by nuclear run-off analysis,Northern blot analysis, or by measuring levels of a gene controlled bythe promoter. The immediate early promoter may be used to control theexpression of fos or jun or any detectable gene product, including, butnot limited to, any of the known reporter genes, such as a gene thatconfers hygromycin resistance (Murphy and Efstratiadis, 1987, Proc.Natl. Acad. Sci. U.S.A. 84:8277-8281) chloramphenicol acetyltransferase(CAT), neomycin phosphotransferase (neo), beta-galactosidasebeta-glucuronidase, beta-galactosidase, etc. of detecting or measuringneurotrophin activity.

Furthermore, the cells used in the assay systems of the invention may ormay not be cells of the nervous system. For example, in a specific,nonlimiting embodiment of the invention, growth-factor dependentfibroblasts may be used as the basis for a signal transducing assaysystem. A fibroblast cell line that is growth factor dependent inserum-free media (e.g. as described in Zham and Goldfarb, 1986, Mol.Cell. Biol. 6:3541-3544) may be transfected with a receptor-encodinggene, for instance by using a CaPO₄ transfection protocol with 5micrograms of DNA of CMV-promoter-based expression vector comprising themusk gene and one microgram of hygromycin-resistance gene-containingexpression vector. After about 48 hours, the cells may then be selectedfor hygromycin resistance to identify positive transfectants. The cellsmay then be cultured for about three weeks in the presence ofhygromycin,and then resistant colonies may be pooled. These cells maythen be plated on tissue culture plates coated with poly-D-lysine andhuman fibronectin, and allowed to grow in DMEM plus 10% bovine calfserum for about four hours to allow the cells to bind to the plates. Theserum-containing media may then be aspirated and the cells may be washedabout three times with PBS to remove any residual serum. The cells maythen be taken up with either serum free defined media (a 3:1 mixture ofDMEM and Hams F12, supplemented with 8 mM sodium bicarbonate, 15 mMHEPES, 4×10⁻⁶M MnCl₂, 3 mM histidine, 10⁻⁵M ethanolamine, 10⁻⁷M sodiumselenite, 5 mg transferrin per liter, 200 mg bovine serumalbumin-linoleic acid complex per liter gentamicin, penicillin, andstreptomycin, 20 mM L-glutamine). Cells produced in this manner, thenincubated with a factor capable of binding to MuSK may, after about 5days in culture (replacing media and growth factors every 48 hours), beexpected to be growing and proliferating; cells treated with anunrelated ligand at 100 ng/ml or in serum free-medium should not,however, proliferate.

Further insight into the physiological role of MuSK will come from thefurther definition of the activating molecule of the present invention.The kinase domain of the MuSK receptor appears to be related to otherreceptor tyrosine kinases, thus it is likely that the MuSK receptor isinvolved in signal transduction in cells in which it is expressed.Accordingly, the MuSK activating molecule of the present invention maybe used to induce signal transduction not only in naturally occurringMuSK-expressing cells, which include cells found in the muscle tissue,heart, spleen, ovaries and retina, but also in cells engineered toexpress the MuSK receptor. The MuSK activating molecule of the presentinvention may be used to promote the growth or survival of such cells.

The term “MuSK activating molecule” as used herein refers to a moleculewhich is capable of inducing phosphorylation of the MuSK receptor in thecontext of a differentiated muscle cell. One such activating molecule isagrin as described in the Examples set forth herein.

As used herein, the term “MuSK activating molecule” includes theisolated and purified MuSK receptor activating polypeptides describedherein, as well as functionally equivalent molecules in which amino acidresidues are substituted for residues within the sequence resulting in asilent change. For example, one or more amino acid residues within thesequence can be substituted by another amino acid of a similar polaritywhich acts as a functional equivalent, resulting in a silent alteration.Substitutes for an amino acid within the sequence may be selected fromother members of the class to which the amino acid belongs. For example,the nonpolar (hydrophobic) amino acids include alanine, leucine,isoleucine, valine, proline, phenylalanine, tryptophan and methionine.The polar neutral amino acids include glycine, serine, threonine,cysteine, tyrosine, asparagine, and glutamine. The positively charged(basic) amino acids include arginine, lysine and histidine. Thenegatively charged (acidic) amino acids include aspartic acid andglutamic acid. Also included within the scope of the invention areproteins or fragments (portions) or derivatives thereof which exhibitthe same or similar biological activity and derivatives which aredifferentially modified during or after translation, e.g., byglycosylation, proteolytic cleavage, linkage to an antibody molecule orother cellular ligand, etc.

The present invention also provides for use of the MuSK receptor or itsextracellular or intracellular domain to screen for drugs that interactwith or activate MuSK. Novel agents that bind to and/or activate thereceptor described herein may mediate survival, proliferation anddifferentiation in cells naturally expressing the receptor, but also maymediate survival, proliferation or differentiation when used to treatcells engineered to express the receptor.

In particular embodiments, the extracellular domain (soluble receptor)of MuSK is utilized in screens for cognate ligands and activatingmolecules. For example, the MuSK receptor activating molecule describedherein may be used in a competition assay to identify agents capable ofacting as receptor agonists or antagonists by competing the agents withMuSK activating molecule for phosphorylation of the MuSK receptor.Specifically, the active portion of human agrin described herein may beused as the MuSK activating molecule in a competition assay to screenfor agents capable of acting as receptor agonists or antagonists.

The present invention also provides for nucleic acid probes, capable ofhybridizing with a sequence included within the nucleotide sequenceencoding human MuSK or its activating molecule, useful for the detectionof MuSK expressing tissue or MuSK activating molecule-expressing tissuein humans and animals. The invention further provides for antibodiescapable of specifically binding MuSK or MuSK activating molecule. Theantibodies may be polyclonal or monoclonal.

The present invention also has diagnostic and therapeutic utilities. Inparticular embodiments of the invention, methods of detectingaberrancies in the function or expression of the receptor describedherein may be used in the diagnosis of muscular or other disorders. Inother embodiments, manipulation of the receptor, agonists which bindthis receptor, or receptor activating molecules may be used in thetreatment of neurological diseases or diseases of muscle orneuromuscular unit disorders, including, but not limited to, musculardystrophy and muscle atrophy. In further embodiments, the extracellulardomain of the receptor is utilized as a blocking agent.

The present invention also provides for an isolated and purifiedpolypeptide which activates MuSK receptor. In one embodiment, thepolypeptide of the invention is encoded by a nucleotide sequencecomprising the coding region of the active portion of human agrincontained in the vector designated as pBluescript human Agrin-1(pBL-hAgrin1) that was deposited with the American Type CultureCollection on Dec. 12, 1995 under ATCC Accession No. 97378. The presentinvention further provides for an isolated polypeptide which isfunctionally equivalent to this polypeptide.

The invention further provides for an isolated and purified nucleic acidmolecule comprising a nucleotide sequence encoding the active portion ofhuman agrin, wherein the nucleotide sequence is selected from the groupconsisting of:

-   (a) the nucleotide sequence comprising the coding region of the    active portion of human agrin contained in the vector designated as    pBL-hAgrin 1 (ATCC Accession No. 97378);-   (b) a nucleotide sequence that hybridizes under stringent conditions    to the nucleotide sequence of (a) and which encodes the active    portion of human a grin; and-   (c) a nucleotide sequence that, as a result of the degeneracy of the    genetic code, differs from the nucleotide sequence of (a) or (b) and    which encodes the active portion of human agrin.

The invention also provides for the above-described nucleic acidmolecule which additionally contains a nucleotide sequence so that theencoded polypeptide contains the eight amino acids ELANEIPV at theposition corresponding to amino acid position 1780 as shown in FIGS.14A-14C (SEQ ID NO: 34).

The invention further provides for an isolated and purified nucleic acidmolecule comprising a nucleotide sequence encoding the active portion ofhuman agrin, wherein the nucleotide sequence is selected from the groupconsisting of:

-   -   (a) the nucleotide sequence comprising the coding region of the        active portion of human agrin as set forth in FIGS. 15A-15B (SEQ        ID NO: 35);    -   (b) a nucleotide sequence that hybridizes under stringent        conditions to the nucleotide sequence of (a) and which encodes        the active portion of human agrin; and    -   (c) a nucleotide sequence that, as a result of the degeneracy of        the genetic code, differs from the micleotide sequence of (a)        or (b) and which encodes the active portion of human agrin.

The invention further provides for an isolated nucleic acid moleculecomprising a nucleotide sequence encoding the active portion of humanagrin, wherein the nucleotide sequence is selected from the groupconsisting of:

-   -   (a) the nucleotide sequence as set forth in FIGS. 15A-15B (SEQ        ID NO: 35);    -   (b) the nucleotide sequence encoding amino acids 24 to 492 as        set forth in FIGS. 15A-15B (SEQ ID NO: 35);    -   (c) the nucleotide sequence encoding amino acids 60 to 492 as        set forth in FIGS. 15A-15B (SEQ ID NO: 35);    -   (d) the nucleotide sequence encoding amino acids 76 to 492 as        set forth in FIGS. 15A-15B (SEQ ID NO: 35);    -   (e) the nucleotide sequence encoding amino acids 126 to 492 as        set forth in FIGS. 15A-15B (SEQ ID NO: 35);    -   (f) the nucleotide sequence encoding amino acids 178 to 492 as        set forth in FIGS. 15A-15B (SEQ ID NO: 35);    -   (g) the nucleotide sequence encoding amino acids 222 to 492 as        set forth in FIGS. 15A-15B (SEQ ID NO: 35);    -   (h) the nucleotide sequence encoding amino acids 260 to 492 as        set forth in FIGS. 15A-15B (SEQ ID NO: 35);    -   (i) the nucleotide sequence encoding amino acids 300 to 492 as        set forth in FIGS. 15A-15B (SEQ ID NO: 35);    -   (j) a nucleotide sequence that hybridizes under stringent        conditions to any of the nucleotide sequences of (a) through (i)        and which encodes the active portion of human agrin; and    -   (k) a nucleotide sequence that, as a result of the degeneracy of        the genetic code, differs from any of the nucleotide sequences        of (a) through (j) and which encodes the active portion of human        agrin.

A further embodiment of the invention is an isolated and purifiednucleic acid molecule encoding agrin 0-8 comprising a nucleotidesequence encoding the active portion of human agrin, wherein thenucleotide sequence is as set forth in FIGS. 15A-15B (SEQ D NO: 35) withthe exception that there is no insert at position Y. Another embodimentof the invention is an isolated and purified nucleic acid moleculeencoding agrin 4-0 comprising a nucleotide sequence encoding the activeportion of human agrin, wherein the nucleotide sequence is as set forthin FIGS. 15A-15B (SEQ ID NO: 35) with the exception that there is noinsert at position Z.

The present invention provides for an isolated polypeptide encoded byany one of the nucleic acid molecules of the invention as set forthherein. Furthermore, the present invention provides for saidpolypeptides modified by covalent attachment of a polyethylene glycolmolecule.

Thus, the present invention provides truncated forms of the human agrinpolypeptide which retain one or more of the biological activities ofhuman agrin. As set forth herein, the invention also provides nucleicacid sequences encoding such truncated forms. These truncated formsretain, for example, the ability to induce phosphorylation of the MuSKreceptor. The truncated forms may be of any suitable length, as long asthey retain one or more of the biological activities of human agrin.Truncated forms including the C-terminal of human agrin are preferred.

Referring to FIGS. 15A-15B (SEQ ID NO: 35), starling at the N-terminalend (amino acid 24—KSPC) these truncated forms of human agrin preferablyhave deletions of up to 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350or 400 amino acids. Particularly preferred truncated forms are describedherein as delta 3 through delta 9.

The invention also provides for a method of promoting the growth orsurvival of a MuSK receptor expressing cell in culture comprisingadministering to the MuSK receptor expressing cell an effective amountof agrin or a derivative of agrin. In one embodiment of this method, theagrin is human agrin. In another embodiment of this method, the MuSKreceptor expressing cell is a cell which is normally found in the heart,spleen, ovary or retina. In another embodiment, the MuSK receptorexpressing cell is a cell which has been genetically engineered toexpress the MuSK receptor.

The present invention also includes a method of treating a patientsuffering from a muscle disease or neuromuscular disorder comprisingadministering to the patient an effective amount of agrin or aderivative thereof. By way of non-limiting example, the agrin may behuman agrin and the derivative may be the active portion of the humanagrin molecule. The active portion of the human agrin molecule may beany one of the truncated fragments' (portions) of human agrin asdescribed herein that is capable of inducing phosphorylation of the MuSKreceptor.

The present invention also includes a method of treating a patientsuffering from a muscle disease or neuromuscular disorder comprisingadministering to the patient an effective amount of agrin or a portionor derivative thereof in combination with Ciliary Neurotrophic Factor(CNTF) or Modified Ciliary Neurotrophic Factor as described in U.S. Pat.No. 5,349,056 issued Sep. 20, 1994 to Panayotatos.

The present invention also includes an antibody capable of specificallybinding human agrin. More specifically, the invention includes anantibody capable of specifically binding the active portion of humanagrin. The antibody may be monoclonal or polyclonal. The inventionfurther provides a method of detecting the presence of human agrin in asample comprising:

-   -   a) reacting the sample with an antibody capable of specifically        binding human agrin under conditions whereby the antibody binds        to human agrin present in the sample; and    -   b) detecting the bound antibody, thereby detecting the presence        of human agrin in the sample.

The antibody used may be monoclonal or polyclonal. The sample may bebiological tissue or body fluid. The biological tissue may be brain,muscle, or spinal cord. The body fluid may be cerebrospinal fluid,urine, saliva, blood, or a blood fraction such as serum or plasma.

The cDNA clone encoding the active portion of human agrin describedherein will facilitate screening of cDNA and genomic libraries in orderto clone the full length sequence coding for the entire human agrinmolecule. Cells may be genetically engineered to produce the activeportion or the full length agrin molecule by, e.g., transfection,transduction, electroporation, microinjection, via a transgenic animal,of a nucleotide sequence encoding the active portion or the full lengthagrin molecule in a suitable expression vector. The invention alsoprovides for a vector comprising an isolated nucleic acid moleculeencoding an active portion or the full length human agrin molecule.

The invention further provides for a host-vector system for theproduction in a suitable host cell of a polypeptide having thebiological activity of human agrin. The suitable host cell may be abacterial cell such as E. coli., a yeast cell such as Pichia pastoris,an insect cell such as Spodoptera frugiperda or a mammalian cell such asa COS or CHO cell. The invention also provides for a method of producinga polypeptide having the biological activity of human agrin whichcomprises growing cells of the host-vector system under conditionspermitting production of the polypeptide and recovering the polypeptideso produced.

The invention further provides for an expression vector comprising anucleic acid molecule encoding human agrin or a portion thereof, whereinthe nucleic acid molecule is operatively linked to an expression controlsequence. The invention also provides a host-vector system for theproduction of a polypeptide having the biological activity of humanagrin which comprises the expression vector of the invention in asuitable host cell. The suitable host cell may be a bacterial cell suchas E. coli., a yeast cell such as Pichia pastoris, an insect cell suchas Spodoptera frugiperda or a mammalian cell such as a COS or CHO cell.The invention further provides for a method of producing a polypeptidehaving the biological activity of human agrin which comprises growingcells of the host-vector system of the invention, under conditionspermitting production of the polypeptide and recovering the polypeptideso produced.

As described above, the present invention relates to a tyrosine kinasereceptor that appears to be expressed in denervated muscle. According tothe present invention, probes capable of recognizing these receptors maybe used to identify diseases or disorders by measuring altered levels ofthe receptor in cells and tissues. Such diseases or disorders may, inturn, be treatable using the activating molecule disclosed herein. Suchdisorders include but are not limited to those in which atrophic ordystrophic change of muscle is the fundamental pathological finding. Forexample, muscle atrophy can result from denervation (loss of contact bythe muscle with its nerve) due to nerve trauma; degenerative, metabolicor inflammatory neuropathy (e.g. Guillian-Barre syndrome), peripheralneuropathy, or damage to nerves caused by environmental toxins or drugs.In another embodiment, the muscle atrophy results from denervation dueto a motor neuronopathy. Such motor neuronopathies include, but are notlimited to: adult motor neuron disease, including Amyotrophic LateralSclerosis (ALS or Lou Gehrig's disease); infantile and juvenile spinalmuscular atrophies, and autoimmune motor neuropathy with multifocalconduction block. In another embodiment, the muscle atrophy results fromchronic disuse. Such disuse atrophy may stem from conditions including,but not limited to: paralysis due to stroke, spinal cord injury;skeletal immobilization due to trauma (such as fracture, sprain ordislocation) or prolonged bed rest. In yet another embodiment, themuscle atrophy results from metabolic stress or nutritionalinsufficiency, including, but not limited to, the cachexia of cancer andother chronic illnesses, fasting or rhabdomyolysis, endocrine disorderssuch as, but not limited to, disorders of the thyroid gland anddiabetes. The muscle atrophy can also be due to a muscular dystrophysyndrome, including but not limited to the Duchenne, Becker, myotonic,Fascioscapulohumeral, Emery-Dreifuss, oculopharyngeal, scapulohumeral,limb girdle, and congenital types, and the dystrophy known as HereditaryDistal Myopathy. In a further embodiment, the muscle atrophy is due to acongenital myopathy, including, but not limited to Benign CongenitalHypotonia, Central Core disease, Nemaline Myopathy, and Myotubular(centronuclear) myopathy. In addition, MuSK and its associated ligandmay be of use in the treatment of acquired (toxic or inflammatory)myopathies. Myopathies which occur as a consequence of an inflammatorydisease of muscle, include, but not limited to polymyositis anddermatomyositis. Toxic myopathies may be due to agents, including, butare not limited to adiodarone, chloroquine, clofibrate, colchicine,doxorubicin, ethanol, hydroxychloroquine, organophosphates,perihexiline, and vincristine.

Although not wishing to be bound by theory, preliminary mapping of muskin the mouse has revealed that the gene is localized to mouse chromosome4 in a region of homology with human chromosome 9q. Mutations in micethat are associated with this region of chromosome 4 include the “wi”mutation (whirler), which results in symptoms of the shaker syndrome,including deafness, head-tossing, circling and hyperactivity (Lane, P.W., 963, J. Hered. 54:263-266). Another mutation in mice that isassociated with this region of chromosome 4 is the “vc” mutation(vacillans) which is associated with the symptoms of violent tremor whenwalking and with swaying of the hindquarters (Sirlin, J. L., 1956, J.Genet. 54:42-48).

In humans, the disease known as idiopathic torsion dystonia (ITD) isassociated with a gene that has been mapped, through linkage analysis tohuman chromosome 9q band 34. This disease is characterized by sustained,involuntary muscle contractions, frequently causing twisting andrepetitive movements or abnormal postures.

Assuming a defect in musk to be associated with these diseases, thepresent invention may be used in gene therapy for the replacement ofsuch gene in situ. Alternatively, probes utilizing a unique segment ofthe musk gene may prove useful as a diagnostic for such disorders. Thepresent invention may also be used, where indicated, in gene therapy forthe replacement of the human agrin gene in situ.

Any of the methods known to one skilled in the art of transferring genesinto skeletal muscle tissue may be used in MuSK or agrin gene therapyprotocols. By way of non-limiting example, one skilled in the art mayutilize direct injection of naked DNA into muscle tissue,adenovirus-associated gene transfer, primary myoblast transplantation,or cationic liposome: DNA complex gene transfer.

For example, direct injection of DNA into muscle may be employed tooptimize vector construction as described by Manthorpe et al., (1993,Hum. Gene Ther. 4: 419-431) in which covalently closed circular plasmidDNA encoding the firefly luciferase reporter gene was injected intoadult murine skeletal muscle for the purpose of evaluating the efficacyof various regulatory elements contained in the DNA expression vector.In a biological study, the systemic immunological effects of cytokinegenes were evaluated using direct injection into muscle of DNA encodingthe genes for IL-2, IL-4, or TGF-β-1 (Raz, et al., 1993, Proc. Natl.Acad. Sci. USA 90: 4523-7). Another study tested the ability of thehuman kallikrein gene product to reduce blood pressure in spontaneouslyhypertensive rats following direct DNA injection of the human kallikreingene into murine skeletal muscle (Xiong, et al., 1995, Hypertension 25:715-719). In studies aimed at evaluating the expression ofmuscle-specific proteins following direct injection of DNA variousdeletion-containing dystrophin gene mutant DNA constructs were injectedinto mdx mouse skeletal muscle and expression patterns andcolocalization studies were performed to investigate dystrophin function(Fritz, et al., 1995, Pediatr. Res. 37: 693-700). Many investigatorsbelieve that it may be not only important but advantageous to regulatethe timing and level of gene expression following gene transfer throughdirect DNA injection. For example, Dhawan et al., (1995, Somat. Cell.Mol. Genet. 21: 233-240) have tested a tetracycline-responsive promotersystem in which orally or parenterally administered tetracycline canregulate reporter gene expression in mouse skeletal muscle followingdirect injection of DNA.

Adenovirus-mediated in vivo gene transfer has been studied extensivelyas a possible method for delivering genes for gene therapy. Recombinantadenovirus vectors containing exogenous genes for transfer are derivedfrom adenovirus type 5 and are rendered replication-deficient bydeletion of the E1 region of the viral genome (Brody & Crystal, 1994,Ann. N. Y. Acad. Sci. 716: 90-101). Huard et al., (1995, Gene Ther.2:107-115) have evaluated the efficiency of viral transduction into rattissues following various routes of administration (intra-arterial,intravenous, gastric-rectal, intraperitoneal, and intracardiac). Theinvestigators report that route of administration is a major determinantof the transduction efficiency of rat tissue by adenovirus recombinants.In addition to route of administration preferences, it has been shownthat vectors carrying U3 region viral long terminal repeats (LTRs)modified in the enhancer region may be used to target tissue- anddifferentiation-specific gene expression into skeletal muscle (Ferrariet al., 1995, Hum. Gene Ther. 6: 733-742). Many studies have beenperformed (Ragot, et al., 1993, Nature 361: 647-50; Petrof, et al.,1995, Am. J. Respir. Cell. Mol. Biol. 13: 508-17; Phelps, et al., 1995,Hum. Mol. Genet 4: 1251-1258; Kochanek, et al., 1996, Proc. Natl. Acad.Sci. USA 93: 5731-36) which have tested adenovirus-mediated transfer offull length and truncated forms of the dystrophin gene into muscle ofnormal and mdx mice.

Transplantation into skeletal muscle tissue of retrovirally transformedprimary myoblasts expressing recombinant genes has also been extensivelystudied as a possible approach to gene therapy for muscle as well asnon-muscle diseases. It is known that the success of myoblasttransplantation for correction of intrinsic muscle defects is dependenton the ability of the transplanted myoblasts to fuse to the hostmyofibers. To address this issue, Rando & Blau (1994, J. Cell. Biol.125:1275-87) developed a novel culture system for isolating enriched andclonal populations of primary myoblasts. Myoblasts isolated by thistechnique were shown to efficiently fuse to host myofibers to formhybrid myofibers persisting for up to six months as evidenced byβ-galactosidase reporter gene expression. Because immunorejection oftransplanted myoblasts is a potential problem in this gene therapyapproach, it has been addressed in studies comparing autologous versusheterologous myoblasts for transplantation (Huard, et al., 1994, Hum.Gene Ther. 5: 949-58) and with Cyclosporin A-induced immunosuppressionin adult mice receiving myoblast transplants following muscle injury(Irintchev et al., 1995, J. Neurocytol. 24: 319-331). Representativedisease targets for gene therapy using myoblast transplantation includehemophilia B in which circulating human or canine factor IX has beenmeasured in the plasma of mice following transplantation of recombinantmyoblasts into skeletal muscles of normal and SCID mice (Roman, et al.,1992, Somat. Cell. Mol. Genet. 18: 247-58; Dai, et al., 1992, Proc.Natl. Acad. Sci. USA 89: 10892-5; Yao, et al., Proc. Natl. Acad. Sci.USA 89: 3357-61; Yao, et al., 1994, Gene Ther. 1: 99-107) and primarymyopathies such as Duchenne muscular dystrophy where myoblastsexpressing the dystrophin gene have been transplanted into normal andmdx mice (Partridge, et al., Nature 337: 176-9; Sopper, et al., 1994,Gene Ther. 1: 108-113).

Plasmid DNA complexed with cationic lipids has been evaluated for itsability to deliver genes into muscle tissue as well. Trivedi, et al.,(1995, J. Neurochem. 64: 2230-38) carried out in vitro studies thatutilized polycationic liposomes to successfully deliver the reportergene LacZ into the cultured mouse myoblast cell line C2C12 and intoprimary mouse myoblasts derived from normal and mdx mice, forming thebasis for adaptation to in vivo gene therapy.

The present invention provides for a method of diagnosing a neurologicalor other disorder in a patient comprising comparing the levels ofexpression of MuSK in a patient sample with the levels of expression ofMuSK in a comparable sample from a healthy person, in which a differencein the levels of expression of MuSK in the patient compared to thehealthy person indicates that a disorder in the patient may be primarilyor secondarily related to MuSK metabolism. A patient sample may be anycell, tissue, or body fluid but is preferably muscle tissue,cerebrospinal fluid, blood, or a blood fraction such as serum or plasma.

One variety of probe which may be used is anti-MuSK antibody orfragments thereof containing the binding domain of the antibody.

According to the invention, MuSK protein, or fragments or derivativesthereof, may be used as an immunogen to generate anti-MuSK antibodies.By providing for the production of relatively abundant amounts of MuSKprotein using recombinant techniques for protein synthesis (based uponthe MuSK nucleotide sequences of the invention), the problem of limitedquantities of MuSK has been obviated.

To further improve the likelihood of producing an anti-MuSK immuneresponse, the amino acid sequence of MuSK may be analyzed in order toidentify portions of the molecule which may be associated with increasedimmunogenicity. For example, the amino acid sequence may be subjected tocomputer analysis to identify surface epitopes which presentcomputer-generated plots of hydrophilicity, surface probability,flexibility, antigenic index, amphiphilic helix, amphiphilic sheet, andsecondary structure of MuSK. Alternatively, the deduced amino acidsequences of MuSK from different species could be compared, andrelatively non-homologous regions identified; these non-homologousregions would be more likely to be immunogenic across various species.

For preparation of monoclonal antibodies directed toward MuSK, or itsactivating molecule, any technique which provides for the production ofantibody molecules by continuous cell lines in culture may be used. Forexample, the hybridoma technique originally developed by Kohler andMilstein (1975, Nature 256:495-497), as well as the trioma technique,the human B-cell hybridoma technique (Kozbor et al., 1983, ImmunologyToday 4:72), and the EBV-hybridoma technique to produce human monoclonalantibodies (Cole et al., 1985, in “Monoclonal Antibodies and CancerTherapy,” Alan R. Liss, Inc. pp. 77-96) and the like are within thescope of the present invention.

The monoclonal antibodies for therapeutic use may be human monoclonalantibodies or chimeric human-mouse (or other species) monoclonalantibodies. Human monoclonal antibodies may be made by any of numeroustechniques known in the art (e.g., Teng et al., 1983, Proc. Natl. Acad.Sci. U.S.A. 80:7308-7312; Kozbor et al., 1983, Immunology Today 4:72-79;Olsson et al., 1982, Meth. Enzymol. 92:3-16). Chimeric antibodymolecules may be prepared containing a mouse antigen-binding domain withhuman constant regions (Morrison et al., 1984, Proc. Natl. Acad. Sci.U.S.A. 81:6851, Takeda et al., 1985, Nature 314:452).

Various procedures known in the art may be used for the production ofpolyclonal antibodies to epitopes of MuSK. For the production ofantibody, various host animals can be immunized by injection with MuSKprotein, or a fragment or derivative thereof, including but not limitedto rabbits, mice, rats, etc. Various adjuvants may be used to increasethe immunological response, depending on the host species, and includingbut not limited to Freund's (complete and incomplete), mineral gels suchas aluminum hydroxide, surface active substances such as lysolecithin,pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanins, dinitrophenol, and potentially useful human adjuvants suchas BCG (Bacille Calmette-Guerin) and Corynebacterium parvum.

A molecular clone of an antibody to a MuSK epitope can be prepared byknown techniques. Recombinant DNA methodology (see e.g., Maniatis etal., 1982, Molecular Cloning, A Laboratory Manual, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.) may be used to constructnucleotide sequences which encode a monoclonal antibody molecule, orantigen binding region thereof.

Antibody molecules may be purified by known techniques, e.g.,immunoabsorption or immunoaffinity chromatography, chromatographicmethods such as HPLC (high performance liquid chromatography), or acombination thereof, etc.

The present invention provides for antibody molecules as well asfragments of such antibody molecules. Antibody fragments which containthe idiotype of the molecule can be generated by known techniques. Forexample, such fragments include but are not limited to: the F(ab′)₂fragment which can be produced by pepsin digestion of the antibodymolecule; the Fab′ fragments which can be generated by reducing thedisulfide bridges of the F(ab′)₂ fragment, and the Fab fragments whichcan be generated by treating the antibody molecule with papain and areducing agent.

The above mentioned probes may be used experimentally to identify cellsor tissues which hitherto had not been shown to express musk.Furthermore, these methods may be used to identify the expression ofmusk by aberrant tissues, such as malignancies. In additionalembodiments, these methods may be used diagnostically to compare theexpression of musk in cells, fluids, or tissue from a patient sufferingfrom a disorder with comparable cells, fluid, or tissue from a healthyperson. Fluid is construed to refer to any body fluid, but particularlyblood, including blood fractions such as serum or plasma, orcerebrospinal fluid. A difference in the levels of expression of musk inthe patient compared to a healthy person may indicate that the patient'sdisorder may be primarily or secondarily related to MuSK metabolism. Anincrease in levels of MuSK, for example, could either indicate that thepatient's disorder is associated with an increased sensitivity to normallevels of MuSK-binding ligand or, alternatively, may suggest that thepatient's MuSK-binding ligand levels are low such that the number ofreceptors is increased by way of compensation.

The present invention further provides for the use of soluble receptor(the extracellular domain) to counter the effect of ligand on MuSKexpressing cells.

EXAMPLE 1 Cloning of the cDNA Encoding MuSK

Tyrosine kinase homology domains were identified based on the alignmentsby Hanks et al. (1988) Science 241, 42-52. Highly conserved regionsAsp-Leu-Ala-Ala-Arg-Asn (SEQ ID NO: 7) AND Asp-Val-Trp-Ser-Tyr-Gly (SEQID NO: 13) were used in designing the following degenerateoligonucleotide primers:

-   5′-TCTTGACTCGAGAYYTNGCNGCNMGNAA-3′ (SEQ ID NO: 8)-   5′-GAATTCGAGCTCCCRTANSWCCANACRTC-3′ (SEQ ID NO: 15)    with which to prime PCR reactions using denervated muscle cDNAs.    Resulting amplified DNA fragments were cloned by insertion into    plasmids, sequenced and the DNA sequences were compared with those    of all known tyrosine kinases. cDNA templates were generated by    reverse transcription of denervated muscle tissue RNAs using oligo    d(T) primers. PCR reactions were done at primer annealing    temperatures of 40° C. Aliquots of the PCR reactions were subjected    to electrophoresis on an agarose gel.

Size-selected amplified DNA fragments from these PCR reactions werecloned into plasmids as follows: Each PCR reaction was reamplified asdescribed above, digested with XhoI and SacI to cleave sites in thetermini of the primers (see below). XhoI/SacI-cut DNAs were purified byMagic PCR kit (from Promega) and cloned into compatible XhoI/SacI sitesin the Bluescript II SK(+) plasmid, introduced into DH10B E. coli byelectroporation, followed by plating of transformants on selective agar.Ampicillin-resistant bacterial colonies from PCR transformation wereinoculated into 96-well microtiter plates and used for PCR using vectorprimers (T3 and T7) flanking the tyrosine kinase insert and these PCRfragments were analyzed by sequencing.

One of the cloned fragment sequences contained a segment of a noveltyrosine kinase domain, which was designated as MuSK. The sequence ofthe PCR-derived fragment corresponding to MuSK was used to generate PCRprimers to obtain longer MuSK specific fragments by the RACE procedure.These longer MuSK probes were used as a hybridization probe to obtainfull length MuSK cDNA clones from a rat denervated skeletal muscle cDNAlibrary. DNA was sequenced by using the ABI 373A DNA sequencer and TaqDyedeoxy Terminator Cycle Sequencing Kit (Applied Biosystems, Inc.,Foster City, Calif.). The sequence of MuSK (FIGS. 1A-1D) has a highdegree of homology to members of the trk family of proteins. It was alsofound to be similar to the Jennings, et al. Torpedo RTK found in muscle.

Oligonucleotide primers corresponding to conserved regions of knowntyrosine kinase molecules were used to amplify and clone DNA sequencesencoding novel orphan tyrosine kinase receptor molecules. The amino acidsequences of representatives from branches of the tyrosine kinase familyand regions of homology within the catalytic domain of these proteinswere used to design degenerate oligonucleotide primers. These primerswere then used to prime PCR reactions using as template a rat denervatedmuscle cDNA library. Resulting amplified DNA fragments were then clonedinto Bluescript II SK(+) plasmid, sequenced, and the DNA sequencescompared with those of known tyrosine kinases. The sequence of a PCRfragment encoding a novel tyrosine kinase named MuSK was used to obtainmore adjoining DNA sequence. A DNA fragment containing MuSK sequenceswas used as a probe to obtain a cDNA clone from a denervated skeletalmuscle library. This clone encodes a novel tyrosine kinase receptor witha high degree of homology to members of the trk family of proteins. Itwas also found to be homologous to the Jennings, et al. Torpedo RTK.FIGS. 1A-1D presents the nucleotide sequence (SEQ ID NO: 2) of the muskclone.

EXAMPLE 2 Identification of Additional Tyrosine Kinases

The novel MuSK sequence is used to obtain homology segments amongreceptor tyrosine kinases which can be used in combination with otherhomology segments. For example, an alignment of the Torpedo trk-relatedkinase with MuSK shows the following conserved protein segment:

-   -   Asp-Val-Trp-Ala-Tyr-Gly (SEQ ID NO: 3)

This homology “box” is not present in any other mammalian tyrosinekinase receptor. Degenerated oligonucleotides essentially based on this“box” in combination with either previously known or novel tyrosinekinase homology segments can be used to identify new tyrosine kinasereceptors.

The highly conserved regions between MuSK and Torpedo TRKAsp-Val-Trp-Ala-Tyr-Gly (SEQ ID NO: 3) as well as additional primersbased on known regions of homology, such as SEQ ID NOS. 5, 7, 9 OR 11,are used in designing degenerate oligonucleotide primers with which toprime PCR reactions using cDNAs. cDNA templates are generated by reversetranscription of tissue RNAs using oligo d(T) or other appropriateprimers. Aliquots of the PCR reactions are subjected to electrophoresison an agarose gel. Resulting amplified DNA fragments are cloned byinsertion into plasmids, sequenced and the DNA sequences are comparedwith those of all known tyrosine kinases.

Size-selected amplified DNA fragments from these PCR reactions arecloned into plasmids as follows. Each PCR reaction is reamplified asdescribed above in Example 1, digested with XhoI and SacI to cleavesites in the termini of the primers (see below). XhoI/SacI-cut DNAs arecloned into compatible XhoI/SacI sites in a plasmid, introduced into E.coli by electroporation, followed by plating of transformants onselective agar. Ampicillin-resistant bacterial colonies from PCRtransformation are inoculated into 96-well microtiter plates andindividual colonies from these PCR clones are analyzed by sequencing ofplasmid DNAs that are purified by standard plasmid miniprep procedures.

Cloned fragments containing a segment of a novel tyrosine kinase domainare used as hybridization probes to obtain full length cDNA clones froma cDNA library.

EXAMPLE 3 Tissue Specific Expression of MuSK

A 680 nts fragment, containing the tyrosine kinase domain of MuSK, wasradiolabeled and utilized in Northern analysis of various rat tissuespecific RNAs. The rat tissue specific RNAs were fractionated byelectrophoresis through a 1% agarose-formaldehyde gel followed bycapillary transfer to a nylon membrane with 10×SSC. The RNAs werecross-linked to the membranes by exposure to ultraviolet light andhybridized at 65° C. to the radiolabeled MuSK probe in the presence of0.5M NaPO4 (pH 7), 1% bovine serum albumin (Fraction V, Sigma), 7% SDS,1 mM EDTA and 100 ng/ml sonicated, denatured salmon sperm DNA. Thefilter was washed at 65° C. with 2×SSC, 0.1% SDS and subjected toautoradiography for 5 days with one intensifying screen and X-ray filmat −70° C. Ethidium bromide staining of the gel demonstrated thatequivalent levels of total RNA were being assayed for the differentsamples.

The MuSK probe hybridized strongly in adult rat tissue (FIG. 3) to a 7kb transcript from denervated skeletal muscle, and weakly to normalmuscle, retina, ovary, heart and spleen. Weaker levels of expressioncould also be found in liver, kidney and lung. It also hybridizes weaklyto a shorter MuSK transcript of about 6 kb in brain, spinal cord andcerebellum.

In embryonic tissue (FIG. 2), MuSK transcripts can be found in body,spinal cord, placenta and head at E12 and E 13.

The high expression of MuSK in muscle and neural tissue suggests thatthe present invention may be utilized to treat disorders of the nervoussystem, specifically the wide array of neurological disorders affectingmotor neurons (see discussion, supra) and the neuromuscular junction.Additionally, high expression of MuSK in heart tissue suggests that thepresent invention may be utilized to treat heart disease, and may, forexample, have prophylactic use in preventing muscle loss during orfollowing a cardiac event. (see discussion, supra). Expression of MuSKin retinal tissue suggests that the present invention may be utilized totreat retina related disorders, including but not limited to retinitispigmentosa. Expression of MuSK in ovaries suggests that MuSK or theligand associated with MuSK may be useful in the treatment of diseasesor disorders involving the ovaries. Finally, expression of MuSK inspleen suggests that MuSK or the ligand associated with MuSK may beuseful in the treatment of diseases or disorders involving the spleen.

EXAMPLE 4 Cloning and Expression of MuSK Receptorbody for Affinity-BasedStudy of MuSK Ligand Interactions

An expression construct was created that would yield a secreted proteinconsisting of the entire extracellular portion of the rat MuSK receptorfused to the human immunoglobulin gamma-1 constant region (IgG1 Fc).This fusion protein is called a Dmk or MuSK “receptorbody” (RB), andwould be normally expected to exist as a dimer in solution based onformation of disulfide linkages between individual IgG1 Fc tails. The Fcportion of the MuSK RB was prepared as follows. A DNA fragment encodingthe Fc portion of human IgG1 that spans from the hinge region to thecarboxy-terminus of the protein, was amplified from human placental cDNAby PCR with oligonucleotides corresponding to the published sequence ofhuman IgG1; the resulting DNA fragment was cloned in a plasmid vector.Appropriate DNA restriction fragments from a plasmid encoding MuSKreceptor and from the human IgG1 Fc plasmid were ligated on either sideof a short PCR-derived fragment that was designed so as to fuse,in-frame, the MuSK and human IgG1 Fc protein-coding sequences. Thus, theresulting MuSK ectodomain-Fc fusion protein precisely substituted theIgG1 Fc in place of the region spanning the MuSK transmembrane andcytoplasmic domains. An alternative method of preparing receptorbodiesis described in Goodwin, et. al. Cell 73: 447-456 (1993).

Milligram quantities of MuSK RB were obtained by cloning the MuSK RB DNAfragment into the pVL1393 baculovirus vector and subsequently infectingthe Spodoptera frugiperda SF-21AE insect cell line. Alternatively, thecell line SF-9 (ATCC Accession No. CRL-1711) or the cell lineBTI-TN-5b1-4 may be used. DNA encoding the MuSK RB was cloned as an EcoRI-NotI fragment into the baculovirus transfer plasmid pVL1393. PlasmidDNA purified by cesium chloride density gradient centrifugation wasrecombined into viral DNA by mixing 3 mg of plasmid DNA with 0.5 mg ofBaculo-Gold DNA (Pharminigen), followed by introduction into liposomesusing 30 mg Lipofectin (GIBCO-BRL). DNA-liposome mixtures were added toSF-21AE cells (2×106 cells/60 mm dish) in TMN-FH medium (ModifiedGrace's Insect Cell Medium (GIBCO-BRL) for 5 hours at 27° C., followedby incubation at 27° C. for 5 days in TMN-FH medium supplemented with 5%fetal calf serum. Tissue culture medium was harvested for plaquepurification of recombinant viruses, which was carried out using methodspreviously described (O'Reilly, D. R., L. K. Miller, and V. A. Luckow,Baculovirus Expression Vectors—A Laboratory Manual. 1992, New York: W.H. Freeman) except that the agarose overlay contained 125 mg/mL X-gal(5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside; GIBCO-BRL). After 5days of incubation at 27° C., non-recombinant plaques were scored bypositive chromagenic reaction to the X-gal substrate, and theirpositions marked. Recombinant plaques were then visualized by additionof a second overlay containing 100 mg/mL MTT(3-[4,5-dimethylthiazol-2-yl]2,5,diphenyltetrazolium bromide; Sigma).Putative recombinant virus plaques were picked by plug aspiration, andpurified by multiple rounds of plaque isolation to assure homogeneity.Virus stocks were generated by serial, low-multiplicity passage ofplaque-purified virus. Low passage stocks of one virus clone (vMuSKreceptor body) were produced.

SF-21AE cells were cultured in serum free medium (SF-900 II, Gibco BRL)containing 1×antibiotic/antimycotic solution (Gibco BRL) and 25 mg/LGentamycin (Gibco BRL). Pluronic F-68 was added as a surfactant to afinal concentration of 1 g/L. Cultures (4 L) were raised in a bioreactor(Artisan Cell Station System) for at least three days prior toinfection. Cells were grown at 27° C., with gassing to 50% dissolvedoxygen, at a gas flow rate of 80 mL/min (aeration at a sparge ring).Agitation was by means of a marine impeller at a rate of 100 rpm. Cellswere harvested in mid-logarithmic growth phase (˜2×10⁶ cells per mL),concentrated by centrifugation, and infected with 5 plaque forming unitsof vMuSK Receptor Body per cell. Cells and inoculum were brought to 400mL with fresh medium, and virus was adsorbed for 2 hours at 27° C. in aspinner flask. The culture was then resuspended in a final volume of 8 Lwith fresh serum-free medium, and the cells incubated in the bioreactorusing the previously described conditions.

Culture medium from vMuSK Receptor Body-infected SF21AE cells werecollected by centrifugation (500×g, 10 minutes) at 72 hourspost-infection. Cell supernatants were brought to pH 8 with NaOH. EDTAwas added to a final concentration of 10 mM and the supernatant pH wasreadjusted to 8. Supernatants were filtered (0.45 mm, Millipore) andloaded on a protein A column (protein A sepharose 4 fast flow or HiTrapprotein A, both from Pharmacia). The column was washed with PBScontaining 0.5 M NaCl until the absorbance at 280 nm decreased tobaseline. The column was washed in PBS and eluted with 0.5 M aceticacid. Column fractions were immediately neutralized by eluting intotubes containing 1 M Tris pH 9. The peak fractions containing the MuSKRB were pooled and dialyzed versus PBS. Recombinant Autographacalifornica baculovirus encoding the Dmk (MuSK) RB was designated “vDmkreceptorbody” and deposited with the American Type Culture Collection,12301 Parklawn Drive, Rockville, Md. 20852 on May 16, 1995 under ATCCAccession No. VR-2507.

EXAMPLE 5 Sequencing of Human MuSK Receptor

In order to obtain the full coding sequence of the human MuSK receptor,oligonucleotides based on the rat sequence were utilized as PCR primersto amplify cDNA from a human muscle biopsy. The PCR fragment so producedwas then sequenced and the resulting new sequence corresponded to apartial sequence of the human MuSK receptor. The novel partial humanMuSK receptor sequence was then used to obtain further sequence throughsuccessive rounds of the RACE procedure. (Frohman, M. A. (1990), RACE:Rapid amplification of cDNA ends. in PCR Protocols, Innis, M. A.Gelfand, D. H., Snincky, J. J., and White, T. J. eds. Academic Press.San Diego).

This process was complemented by obtaining human genomic clones of MuSKand using the coding sequence of the genomic MuSK to designoligonucleotide primers used to amplify the biopsy cDNA. Stretches ofthe human MuSK cDNA sequence which were difficult to sequence, absent orpresenting some ambiguity were confirmed, corrected or added from thehuman genomic MuSK sequence. MuSK cDNA variants produced by alternativesplicing of MuSK transcripts may be obtained by using this sequence toobtain MuSK cDNA from human sources. The deduced amino acid sequence ofthe human MuSK receptor and the nucleotide sequence encoding it is setforth in FIGS. 4A-4D (SEQ ID NO: 32). One of skill in the art willreadily recognize that this sequence may be used to clone full length,naturally occurring cDNA sequences encoding the human MuSK receptor,which may vary slightly from the sequence set forth in FIGS. 4A-4D (SEQID NO: 32)

EXAMPLE 6 Homologous Recombination to Disrupt the MuSK Gene

The tyrosine kinase domain of MuSK is comprised of 11 subdomains thatare divided among three coding exons. Subdomain I encoding theATP-binding domain is located on the first kinase exon, while subdomains5-11 encoding the catalytic region are located on the third kinase exon(FIG. 5). To disrupt MuSK tyrosine kinase activity, a targeting vectorwas designed that would delete most of the third kinase exon uponhomologous recombination into the endogenous mouse MuSK locus (FIG. 5);this targeting vector contained a total of 3.8 kb of homology with themouse MuSK gene.

The MuSK gene targeting vector was constructed from mouse genomic DNAfragments isolated from a lambda FIX II phage library prepared with 129strain mouse genomic DNA (Stratagene). The 1.7 kb SpeI fragment depictedin FIG. 5 was ligated into the compatible ends of a unique XbaI siteupstream of the PGK-neo cassette (destroying the SpeI and XbaI sites),while the 2.1 kb BamHI DNA fragment depicted in FIG. 5 was blunt-endligated into the unique HindIII site between the PGK-neo cassette andMC1-tk expression cassettes (destroying the BamHI and HindIII sites).The targeting vector was linearized by digestion with NotI and thenelectroporated into E14.1 embryonic stem cells, which were subjected toa double selection protocol (gancyclovir addition resulted in a 5-10fold enrichment compared with selection in G418 alone) and then used togenerate chimeric mice as previously described (Conover et al., 1995;DeChiara et al., 1995).

Successful gene targeting using this construct was predicted to resultin the generation of a novel 3.8 kb EcoRI fragment from the targetedallele as detected by a 5′ probe, as well as loss of two NcoI fragmentshybridizing to a kinase probe (FIG. 5). Southern blot screening forthese fragments revealed that successful targeting of the mouse MuSKgene was achieved in four of approximately 400 embryonic stem (ES) cellclones obtained using a double selection scheme intended to enhance forselection of targeted clones; the ES cells were derived from the 129strain of mice. Male chimeras derived from all four of these targetedclones were bred with C57BL/6 females. Chimeras from two of the targetedclones transmitted the mutant allele to the F1 generation. The resultingF1 progeny heterozygous for the MuSK mutation were viable and appearednormal and fertile.

EXAMPLE 7 MuSK Gene Disruption Results in Perinatal Lethality

The heterozygous F1 progeny were interbred to generate mice homozygousfor the MuSK gene disruption (designated MuSK−/− mice). Among the F2litters derived from these crosses were newborn mice that diedperinatally. Genotype analysis of tail DNA mice revealed that the deadpups were homozygous for the mutant MuSK allele (FIG. 6); significantly,not a single mouse homozygous for the mutation survived the perinatalperiod (37 homozygotes were noted among the first 138 pups that weregenotyped, corresponding to a 26.8% frequency of homozygotes).

To determine the phenotype of the MuSK−/− newborns immediately at birth,applicants were careful to observe the births of several litters derivedfrom heterozygote crosses. Though normal in their gross anatomy and bodyweight, the MuSK−/− pups differed in several striking ways from theirlittermate controls. First, they showed no spontaneous movement and didnot respond to a mild tail or leg pinch. Only a strong tail pinch wasable to elicit a weak uncoordinated movement. By contrast, littermatecontrols showed extensive movement and responded vigorously to a mildtail pinch. Second, the MuSK−/− pups were cyanotic at birth and appearednot to breathe, although their hearts continued to beat for a short timeafter birth. To determine whether the MuSK−/− pups had ever taken abreath, applicants examined the lungs histologically. Lung alveoli arecollapsed in utero, and expand with the first breath of life; even ifrespiration is then terminated, the alveoli remain expanded.

Histological examination (FIG. 7A) revealed that the alveoli of MuSK−/−pups were not expanded, indicating that the pups had never taken abreath. In contrast, the lungs of the littermate controls displayedexpanded alveoli (FIG. 7B).

EXAMPLE 8 Normal Skeletal Muscle in MuSK−/− Mice

Because MuSK is localized to synaptic sites in skeletal muscle(Valenzuela, D., et al., 1995, Neuron 15: 573-584) and because MuSK−/−mutant mice are immobile at birth and die shortly thereafter, applicantsreasoned that neuromuscular synapse formation might be aberrant inMuSK−/− mutants. Applicants first examined the diaphragm muscle becauseits simple organization and thin structure allows synaptic sites to bevisualized in whole-mount preparations. The diaphragm muscle isinnervated by the phrenic nerve, which normally enters near the centerof the diaphragm muscle. The main intramuscular nerve is orientedperpendicular to the long axis of the muscle fibers and extends throughthe central region of the muscle.

For whole-mount diaphragm preparations newborn mice were fixed in 1%paraformaldehyde in phosphate-buffered saline (PBS) at 4° C. for severalhours and then rinsed briefly in PBS. Diaphragm muscles were dissectedout, washed twice for 10 minutes in PBS, incubated in 0.1M glycine inPBS for 15 minutes, rinsed for 5 minutes in PBS, and permeabilized with0.5% Triton X-100 in PBS (PBT) for 5 minutes. The muscles were thenincubated with rabbit antibodies to synaptophysin (kindly provided byDr. R. Jahn, Yale University Medical School), which were diluted 1/1000in PBT with 2% BSA, overnight at 4° C., subsequently rinsed for 5minutes in PBT, washed three times for one hour in PBT and thenincubated, simultaneously with flourescein-conjugated sheep anti-rabbitIgG (Boehringer Mannheim) and tetramethlyrhodamine-conjugatedα-bungarotoxin (α-BGT) (Molecular Probes, Oregon) overnight at 4° C. Thetissues were then washed three times for 1 hour with PBT, rinsed oncewith PBS for 5 minutes, fixed in 100% methanol at −20° C. and mounted in90% glycerol, 0.1 M Tris, pH 7.5 with 1 mg/ml p-phenylenediamine. Thewhole-mounts were viewed with epiflourescence and filters that wereselective for rhodamine or flourescein, and images were recorded eitheron film or on a CCD camera (Princeton Instruments).

The arrangement and gross structure of the muscle fibers (compare FIGS.7C and 7D), as well as of the main intramuscular nerve, appeared to beunaltered in MuSK−/− mutant mice. Thus, although the onset of MuSKexpression occurs at about embryonic day 11 in developing mouse somites(within the presumptive myotome), MuSK does not appear to be essentialfor the generation, proliferation and fusion of myoblasts, or for thegrowth of motor axons from spinal cord to muscle.

EXAMPLE 9 Agrin Fails to Induce AChR Clustering in Myotubes Lacking MuSK

The localization of MuSK to the NMJ inspired us to ask whether MuSK isrequired for responsivity to agrin. To test this, applicants firstisolated myoblasts from newborn MuSK^(−/−) mice or from control pups,attempted to differentiate them into myotubes in culture, and thenassayed for their responsiveness to agrin.

Primary myoblast cultures were established from hind limb musculature ofnewborn MuSK−/− or littermate control pups. This tissue was treatedsequentially with collagenase and trypsin, then plated onto plastictissue culture dishes. After 1 hour, non-adherent cells (principallymyoblasts) were removed and plated onto chamber slides coated withpoly-D-lysine and fibronectin. Myoblast cultures were maintained inDulbecco's Modified Eagle's Medium (DMEM) containing 25% fetal calfserum, 10% horse serum, and 50 μg/ml gentamycin. To induce myotubeformation, cultures were switched to a medium consisting of DMEMcontaining 5% horse serum, L-glutamine and gentamycin to which 20 μMcytosine arabinoside was added after 24 hr. After an additional 2-3days, contractile myotubes had formed abundantly in cultures from bothMuSK−/− and control pups. C2C12 cells were maintained and caused todifferentiate in a serum-poor medium as previously described (Ferns, M.,et al., 1993, Neuron 11: 491-502).

For agrin-mediated AChR clustering assays on primary myotubes, cultureson chamber slides were treated overnight with c-agrin4,8 at 0.01-100 nM;for evaluating MuSK-Fc as an inhibitor of clustering, differentiatedC2C12 cells, on chamber slides coated with fibronectin andpoly-D-lysine, were pretreated with MuSK-Fc or a control receptor-bodyfor 1 hr at 37° C. before addition of approximately 10 nM agrin4,8 forovernight incubation. Following overnight treatments with agrin, thecells were next incubated in rhodamine-conjugated α-bungarotoxin tolabel AChRs, then fixed and mounted for fluorescence microscopy. Toquantify the extent of AChR clustering, randomly selected myotubes wereviewed under fluorescein optics, then switched to rhodamine optics andthe number of AChR clusters within a reticule grid aligned along thelong axis of the myotube were counted. AChRs on the surface of culturedprimary myotubes were quantitated by incubating live cultures with 25mCi 125I-α-BGT for 1 hr at room temperature, washing, and then lysingthe cells in 0.1 N NaOH. The protein concentration in aliquots of eachextract was determined using a BCA protein assay kit (Pierce), while theremainder of the extract was counted in a gamma counter.

Myoblasts from both the control and MuSK^(−/−) mice were able to fuseand form long, twitching myotubes in culture. Together with theobservation that skeletal muscle appears rather normal in MuSK^(−/−)mice, these findings indicate that MuSK is not critical for early muscledevelopment and myoblast fusion. On the other hand, MuSK appeared to beabsolutely required for AChR clustering in response to agrin. Afterstimulation with the most active form of c-agrin, containing both thefour and eight amino acid insertions (c-agrin_(4,8)), AChR clusters wereevident only in the myotubes from control mice (FIG. 8A). While clusterswere induced in normal myotubes with as little as 1 nM c-agrin_(4,8), noclustering was observed in MuSK^(−/−) myotubes even after increasing theconcentration of c-agrin_(4,8) to as high as 100 nM (FIG. 8B). Lack ofdetectable clustering was not due to the absence of AChRs, sincemyotubes from MuSK^(−/−) mice expressed similar numbers of AChR on theirsurface as did myotubes from control mice (FIG. 8C). Thus MuSK appearedto be absolutely required for AChR clustering in response to agrin.

EXAMPLE 10 Agrin Induces Prominent and Rapid Tyrosine Phosphorylation ofMuSK

The inability of agrin to induce AChR clustering in myotubes fromMuSK^(−/−) mice demonstrates that MuSK is required for agrinresponsiveness, and is consistent with the possibility that MuSK servesas the functional agrin receptor. However, since clustering occurs overa period of hours, these results are also consistent with thepossibility that MuSK acts much further downstream in the agrinsignaling pathway. To begin to distinguish between these possibilities,applicants took advantage of the fact that RTKs become rapidlyautophosphorylated on tyrosine upon challenge with their cognate ligand.Applicants decided to assay four of the known forms of solubleagrin—which exhibit differing AChR clustering activities (Ruegg, M. A.et al., 1992, Neuron 8: 691-699; Ferns, M., et al., 1992, Neuron 8:1079-1086; Ferns, M., et al., 1993, Neuron 11: 491-502; Hoch, W. et al.,1994, EMBO J. 13: 2814-2821)—for their ability to induce phosphorylationof the MuSK receptor.

The ability of various agrins and growth factors to induce MuSK or ErbB3tyrosine phosphorylation, for the indicated times and at the indicatedconcentrations, was evaluated in primary rat myoblasts and in eitheruntransfected C2C12 myoblasts, or in C2C12 myoblasts stably transfectedwith a chick MuSK-expressing plasmid. The cells were challenged atconfluence in an undifferentiated state, or approximately 4-5 days afterbeing induced to differentiate into myotubes in serum-poor media. Afterchallenge, the cells were lysed, the extracts subjected toimmunoprecipitation with receptor-specific antibodies, and thenimmunoblotted with either receptor-specific or phosphotyrosine-spedficantibodies, using methods previously described (Stitt, T., et al., 1995,Cell 80: 661-670). Polyclonal antibodies for MuSK were generated asfollows: for rat MuSK, rabbits were immunized with a peptidecorresponding to the carboxy-terminal 20 amino acids of the rat MuSKprotein (Valenzuela, D., et al., 1995, Neuron 15: 573-584; thenomenclature for this antibody is: 41101K); for chick MuSK, rabbits wereimmunized with a peptide corresponding to the first 19 amino acids ofthe chick MuSK cytoplasmic domain (Pep tide: TLPSELLLDRLHPNPMYQ (SEQ IDNO: 16); the nomenclature for this antibody is 52307K). The specificityof the antibodies was determined on Cos-cell expressed MuSK proteins, byboth immune-precipitation and Western, comparing untransfected Cos celllysates to lysates from rat and chicken-MuSK transfected Cos cells.41101K immnune precipitates and Westerns rodent MuSK, but does notrecognize chicken MuSK. 52307 immune precipitates and Westerns chickenMuSK. Antibodies to ErbB3 were obtained from Santa Cruz Biotechnology,Inc.

Cultures of confluent C2C12 cells, either undifferentiated ordifferentiated in serum-poor media for four to five days as describedabove, were transferred to 4° C. and incubated for 90 minutes witheither MuSK-Fc or TrkB-Fc (at 5 mg/ml), each in the presence of theindicated mock or agrin-containing conditioned media (with 100 nMagrin). Agrin levels were determined by Western analysis of theconditioned media with a rat agrin antibody (131, from StressGen, Inc.),using a purified agrin control of known concentration. Following theseincubations, the cells were washed four times with PBS containingcalcium and magnesium, and then incubated for an additional hour withradio-iodinated goat anti-human IgG (NEN/Dupont; 1 mCi/ml in PBS) todetect surface-bound receptor-Fc. After four additional washes, cellswere solubilized in 0.1N NaOH, and bound radioactivity was determined.The assay is similar to that described elsewhere (Davis, S., et al.,1994, Science 266: 816-819).

Transient transfections using either previously described agrinconstructs (Ferns, M., et al., 1993, Neuron 11: 491-502) or empty vectorcontrols, or stable transfections of a chick MuSK-expression construct,were performed as described (Glass, D., et al., 1991, Cell 66: 405-413;Ip, N.Y., et al., 1992, PNAS (USA) 89: 3060-3064). Agrin concentrationsin conditioned media derived from transient transfections were estimatedby immunoblot comparisons with purified agrin of known concentration.

Phosphorylation was assessed on the endogenous MuSK receptor that ishighly expressed in myotube cultures, obtained by differentiating eitherthe C2C12 mouse myoblast cell line (Valenzuela, D., et al., 1995, Neuron15: 573-584) or primary rat myoblasts. Strikingly, soluble agrinscontaining the eight amino acid insert at position Z (c-agrin_(4,8) andc-agrin_(0,8)), which are the forms capable of inducing AChR clustering,were also the forms that induced prominent tyrosine phosphorylation ofMuSK (FIG. 9A). The agrin most active in clustering (c-agrin_(4,8)) wasalso most active in inducing MuSK phosphorylation (FIG. 9A). Incontrast, the soluble agrins lacking the eight amino acid insert(c-agrin_(4,0) and c-agrin_(0,0)), which cannot induce AChR clustering,also could not induce MuSK phosphorylation (FIG. 9A).

The specificity of action of agrin was further explored by comparing itsactivity to growth factors known to have receptors on muscle. Of theseveral such factors tested, including insulin, fibroblast growth factor(FGF) and ARIA/neuregulin, only agrin could induce phosphorylation ofMuSK (FIG. 9A); since FGF also induces AChR clustering on myotubes(Peng, H. B., et al., 1991, Neuron 6: 237-246), these results alsoindicate that MuSK phosphorylation is specific to agrin responses andnot just to agents capable of inducing clustering. Furthermore, whilesuch factors could be shown to induce phosphorylation of their own RTKson myotubes (e.g., neuregulin induces phosphorylation of its cognateRTK, erbB3), agrin could only activate MuSK and not other RTKs (FIG.9B).

The activation of a RTK by its cognate ligand typically tends to occurrapidly, and applicants could demonstrate that agrin induces tyrosinephosphorylation of MuSK with kinetics similar to those seen forwell-characterized RTK/ligand systems (e.g. Kaplan, D. R., et al., 1991,Nature 350: 158-160); induction was detectable by one minute, peakedwithin the first five minutes, and remained elevated for over an hour(FIG. 9D). The tyrosine phosphorylation of MuSK also occurred usingagrin at concentrations similar to those noted for other ligands thatact on RTKs (Ip, N.Y., et al., 1993, Neuron 10: 137-149), withphosphorylation detectable using 1 nM agrin (FIG. 9C).

The requirement of MuSK for agrin responsiveness, the ability of agrinto induce rapid and prominent MuSK phosphorylation, the specificity ofagrin for MuSK as compared to other factors tested, and the precisecorrelation of agrin forms active in AChR clustering assays and in MuSKphosphorylation assays, together continue to support the notion thatMuSK serves as the functional agrin receptor.

EXAMPLE 11 Agrin Does Not Directly Bind to an Isolated MuSK Ectodomain

If MuSK is indeed the functional agrin receptor, applicants would expectto be able to demonstrate binding of agrin to MuSK. In an attempt todemonstrate such binding, applicants first constructed an expressionconstruct encoding a fusion protein between the ectodomain of rat MuSKand the Fc portion of human immunoglobulin G1 (designated MuSK-Fc), andthen produced and purified the fusion protein. Similar receptor-Fcfusions have previously been used to characterize binding between RTKsand their ligands (Davis, S., et al., 1994, Science 266: 816-819; Stitt,T., et al., 1995, Cell 80: 661-670).

Baculovirus expression vectors encoding MuSK-Fc, TrkB-Fc, and Ret-Fcproduced fusion proteins in which the ectodomains of rat TrkB, rat Ret,or rat MuSK, respectively, were linked to a spacer with the sequenceGly-Pro-Gly, followed by the hinge, CH2, and CH3 regions of human IgG1,beginning with the residues Glu-Pro-Lys, as described (Davis, S., etal., 1994, Science 266: 816-819). Baculovirus infections into Spodopterafrugiperda SF-21AE insect cells were performed by standard methods(Stitt, T., et al., 1995, Cell 80: 661-670). The soluble Fc-containingproteins were purified by protein A-Sepharose (Pharmacia)chromatography.

The binding of agrin to immobilized MuSK-Fc as compared to a monoclonalantibody specific for agrin was evaluated by use of BIAcore biosensortechnology (Pharmacia Biosensor), using approaches previously described(Stitt, T., et al., 1995, Cell 80: 661-670). Heparin and CaCl2 weresupplied by Sigma Chemical Co. (St. Louis, Mo.) and used without furtherpurification. The agrin-specific monoclonal antibody (clone AGR131generated to rat agrin) was purchased from StressGen BiotechnologiesCorp. (Victoria, BC, Canada).

In a first approach, applicants used MuSK-Fc together with BIAcorebiosensor technology. The BIAcore technology allows for the direct andquantitative measure of binding of soluble ligands to receptors coupledonto a sensor chip. Recombinant MuSK-Fc was covalently coupled to asurface on the BIAcore sensor chip, and as a control, a monoclonalantibody specific for rat agrin was also coupled to a separate surfaceon the sensor chip; media containing c-agrin_(4,8) was then passed overthe two surfaces. While robust binding of the agrin to the antibodysurface was easily detected, no binding of the agrin to the MuSK surfacecould be seen (FIG. 10A). Furthermore, while binding to the antibodysurface was specifically competable by excess soluble antibody added tothe agrin-containing media, the binding was not competable by excesssoluble MuSK-Fc (FIG. 10A). Since agrin activity requires calcium (Boweand Fallon, 1995, Ann. Rev. Neurosci. 18: 443-462), and because someheparin-binding factors require heparin to bind to their receptors(Goldfarb, M., 1990, Cell Growth & Differentiation 1: 439-445),applicants also attempted binding in the presence of calcium or heparin;in neither case was binding to the MuSK surface observed (FIG. 10A).

Next, applicants tried to demonstrate binding of MuSK and agrin byattempting to use MuSK-Fc to detect agrin immobilized ontonitrocellulose. In contrast to our control experiments, in whichimmobilized brain-derived neurotrophic factor (BDNF) was easily detectedby an Fc fusion of its cognate receptor (TrkB-Fc), and in whichimmobilized agrin was easily detected by the agrin-specific monoclonalantibody, immobilized agrin could not be detected by MuSK-Fc (FIG. 10B).

The negative binding results described above demonstrate that theisolated MuSK receptor is not sufficient to bind agrin. Thus, despitethe plethora of functional data indicating that agrin acts via MuSK,MuSK may not directly serve as a receptor for agrin. Alternatively, MuSKmay require additional components or modifications which are requiredfor it to bind and respond to agrin.

EXAMPLE 12 Agrin Activates MuSK in a Cell-Context-Dependent Fashion:Requirement for a Myotube-Specific Accessory Component

Based on the results described above, applicants considered thepossibility that the agrin-MuSK interaction requires additionalcomponents. To further explore this possibility, applicants determinedthe cell-context dependency for agrin activation of MuSK, reasoning thatif an accessory component was required, it might be specificallyexpressed only on cells normally responding to agrin. Thus applicantsectopically expressed full-length cDNAs encoding rat, human and chickenMuSK in fibroblasts, and assayed for whether these MuSK receptors couldbe inducibly phosphorylated by agrin. When expressed in fibroblasts,none of the three species of MuSK could be phosphorylated in response toagrin. While this supported the possibility that MuSK requires anaccessory myotube-specific component to respond to agrin, it was alsopossible that our cDNAs encoded MuSK variants that could not respond toagrin. This was a potentially worrisome possibility since there aremultiple differently spliced versions of the MuSK transcript(Valenzuela, D., et al., 1995, Neuron 15: 573-584), applicants did notknow which of the forms were normally agrin-responsive, and our cDNAsonly accounted for a subset of the variant forms. Thus applicantsdecided to express our cDNAs in myoblasts to verify that they couldmediate responses to agrin when expressed in the right context. For thispurpose applicants chose to express the chicken MuSK in the mouse C2C12myoblast cell line, since the chicken MuSK could easily be distinguishedfrom the endogenous mouse MuSK based on size and by using particularantibodies. When expressed in undifferentiated myoblasts, the chickenMuSK did not undergo phosphorylation in response to any isoforms ofagrin (FIG. 11, see lanes indicated “Undif”, upper panel), just as itdid not undergo phosphorylation in fibroblasts; undifferentiated C2C12cells do not express appreciable amounts of endogenous MuSK (FIG. 11,lanes indicated “Undif”, lower panel and also (Valenzuela, D., et al.,1995, Neuron 15: 573-584), so applicants could not compare activation ofthe endogenous mouse MuSK in myoblasts. Upon differentiation intomyotubes, the introduced chicken MuSK was as effectively activated byagrin as was the endogenous mouse MuSK (FIG. 11, lanes indicated “Diff”,upper panel); both introduced and endogenous MuSK had identical profilesof responsivity to the various forms of agrins, with activationsmediated only by forms having the eight amino acid insert at the Zposition. Thus our cDNAs encode MuSK proteins that are perfectlycompetent to undergo agrin-induced phosphorylation, but they can only beactivated by agrin in the context of a differentiated myotube,consistent with the notion that agrin activation of MuSK requires amyotube-specific accessory component that is not expressed infibroblasts or undifferentiated myoblasts.

EXAMPLE 13 A Receptor Complex can be Demonstrated Between Agrin, MuSKand a Myotube-Specific Accessory Component

Altogether, the above data indicate that agrin requires MuSK to mediateclustering and that agrin activates MuSK very rapidly, but that agrindoes not directly bind to a purified MuSK ectodomain and can onlyactivate MuSK in the context of a myotube. These findings are consistentwith the possibility that MuSK is a requisite part of an agrin receptorcomplex, but that although MuSK provides a key signaling function forthis complex, it requires another component(s) to bind to agrin. Similartypes of receptor complexes have been described for other ligands.Perhaps some of the best characterized examples include the receptorcomplexes for ciliary neurotrophic factor (CNTF) and its cytokinerelatives (Davis, S., et al., 1993, Science 260: 1805-1808; Stahl andYancopoulos, 1993, Cell 74: 587-590). In order to interact with its twosignal transducing “b” receptor components, gp130 and LIFRb, CNTF mustfirst bind to its “a” receptor component, known as CNTFRa. CNTFRa servesno signaling role, and is in fact linked to the surface via aglycosylphosphatidylinositol linkage and thus has no cytoplasmic domain.The receptor complex for CNTF is built in step-wise fashion: CNTF firstbinds to CNTFRa; this initial complex can then bind to and recruit asingle “b” component; finally, a complete complex forms that involves“b” component dimerization, which is required for signal initiation(FIG. 12A). In the final complex, CNTF seems to make contacts with allthree receptor components. Interestingly, receptor complexes for CNTFcan be built in solution using just the soluble ectodomains of thevarious components. Furthermore, if just one of the receptor componentsis linked to the surface, a receptor complex can be built around itusing soluble versions of the other components, but only in aCNTF-dependent fashion (FIG. 12B).

If agrin binds to MuSK in a receptor complex, applicants reasoned thatthey might be able to manipulate this complex in much the same way theCNTF receptor complex can be manipulated. To explore the possibilitythat myotubes specifically express an accessory component(s) requiredfor agrin to bind MuSK (FIG. 12C), applicants decided to test whetherapplicants could specifically build a receptor complex on the surface ofmyotubes, but not on other cells, using agrin together with a solubleversion of the MuSK receptor to complex to the putative accessorycomponent(s) on the surface of myotubes. Confirming this possibility,applicants found that the binding of soluble MuSK-Fc to the surface ofcells can be increased using agrin, but only on the surface ofdifferentiated myotubes and not on the surface of fibroblasts ormyoblasts (FIG. 13A). These data demonstrate that complexes can formbetween agrin and MuSK, but only in the presence of a myotube-specificcomponent(s) (as suggested in FIG. 12C). Interestingly, although formsof c-agrin containing the eight amino acid insert at the Z position arebest able to promote agrin-dependent MuSK complex formation, forms ofc-agrin without this insert can also form these complexes. The abilityof all the soluble forms to promote complex formation, including thoselacking the eight amino acid insert for activity, may be related toprevious findings that matrix-bound forms of agrins lacking the Z insertcan activate clustering (Ferns, M., et al., 1992, Neuron 8: 1079-1086).Thus although soluble agrins lacking inserts at the Z position do notseem capable of signaling, they may be able to form partial complexes,while matrix-associated forms of these same agrins can proceed to formcomplete signaling-competent complexes. Interestingly, ligands for theEPH family of RTKs provide an example of ligands that bind but do notactivate their receptors when presented in soluble form, but which canact as potent activators when bound to the cell surface (Davis, S., etal., 1994, Science 266: 816-819); deliberate clustering of the solubleligands can allow them to activate as well, suggesting that the role ofsurface-attachment is to allow for ligand-clustering (Davis, S., et al.,1994, Science 266: 816-819).

In the absence of added agrin, the MuSK-Fc exhibited much higher bindingto myotube surfaces than did several control receptor-Fc fusion proteins(FIG. 13A, data shown for TrkB-Fc); the MuSK-Fc, however, displayedsimilar agrin-independent binding to both myoblast and fibroblasts asdid control receptor-Fc proteins (FIG. 13A). Specific binding of MuSK-Fcto myotube surfaces, in the absence of exogenously provided agrin, mayindicate that MuSK has an affinity for its myotube-specific accessorycomponent in the absence of ligand. Alternatively, since myotubes makemuscle forms of agrin (lacking the eight amino acid insert at the Zposition), the specific binding of MuSK-Fc to myotubes in the absence ofadded agrin could be explained by the formation of a complex between theadded MuSK-Fc and endogenously expressed muscle agrin along with theaccessory component; adding additional exogenous soluble agrin maysimply allow for even more MuSK to be recruited into complexes with themyotube-specific accessory component. Although both myoblasts andmyotubes make endogenous agrin, myoblasts seemingly cannot formcomplexes with added MuSK-Fc since they do not express the requiredaccessory component.

To confirm that MuSK directly interacts with agrin as part of itsreceptor complex, applicants next demonstrated that radiolabelled agrincould be cross-linked to MuSK receptors on the surface of myotubes.

Flg-tagged human agrin protein corresponding to the COOH-terminal 50 kDof human agrin 4,8 was expressed in Cos cells and purified by affinityand size-exclusion chromatography to >95% purity. Twenty μg wereiodinated by a modification of the lactoperoxidase method describedpreviously (DiStefano, P., et al, 1992, Neuron 8: 983-993).Incorporation of 125I was greater than 80%; 1251-h-agrin 4,8-flg wasseparated from free 125I on a 1×3 cm Sephadex G-25 column prior to usein cross-linking assays. Specific activity was ˜4000 cpm/fmol (˜2400Ci/mmol). Biological activity of 125I-h-agrin 4,8-flg was monitored bytyrosine phosphorylation of MuSK in C2C12 myotubes and was found to beindistinguishable from its unlabeled counterpart. For cross-linkingstudies, 10 cm plates of differentiated C2C12 myotubes were incubated in1 nM of [125I]-agrin_(4,8) in 1.5 ml of PBS containing 1% BSA and 1mg/ml glucose in the presence or absence of 150-fold excess unlabeledagrin_(4,8) for 75 min at 4° C. The cross-linking agent DSS(disuccinimidyl suberate) was added to a final concentration of 0.2 mMand the plates were incubated at room temperature for 30 min, washed 3times with 50 mM Tris/150 M NaCl pH 7.5, lysed, and subjected toimmunoprecipitation with MuSK-specific antibodies. For peptidecompetition, peptide antigen was included in the immunoprecipitation ata final concentration of 20 μg/ml. The samples were then electrophoresedand the fixed and dried gels were exposed for autoradiography.

Immunoprecipitations using a MuSK-specific antibody, from lysates ofmyotubes chemically cross-linked to radiolabelled recombinant humanagrin contained complexes corresponding in size to agrin/MuSK complexes(FIG. 13B). These agrin/MuSK complexes were not seen in the presence ofexcess unlabelled agrin, or if a peptide was used to block MuSKprecipitation (FIG. 13B). Additional radiolabelled species thatimmunoprecipitated with the MuSK antibody correspond to forms of agrinthat are associated with, but not cross-linked to, MuSK, presumably dueto the low efficiency of cross-linking (FIG. 13B); low levels ofadditional agrin complexes, perhaps involving MASC, could also bedetected in these immunoprecipitations.

Finally, if our findings that soluble MuSK could form complexes with itsrequisite myotube-specific accessory component are correct, then thissoluble receptor should also act as an inhibitor of agrin-mediatedresponses by sequestering the accessory component and preventing it frominteracting with the endogenously-expressed, signaling-competent MuSK.Indeed, addition of increasing amounts of MuSK-Fc did inhibitagrin-mediated clustering of AChRs (FIG. 13C) as well as agrin-inducedMuSK phosphorylation in a dose-dependent manner, while controlreceptor-Fc proteins had no inhibitory effect.

EXAMPLE 14 Cloning of Human Agrin cDNA

Probes corresponding to human agrin were prepared by PCR based onpartial sequences of human agrin available from the Genbank database.

Two pairs of PCR primers were synthesized based on human agrin cDNAsequences obtained from Genbank. The sequences of the oligonucleotideprimers were as follows:

Primer pair 18: h-agrin 18-5′ : 5′-GACGACCTCTTCCGGAATTC-3′ (SEQ ID NO:17) h-agrin 18-3′ : 5′-GTGCACATCCACAATGGC-3′ (SEQ ID NO: 18) Primer pair35: h-agrin 35-5′ : 5′-GAGCAGAGGGAAGGTTCCCTG-3′ (SEQ ID NO: 19) h-agrin35-3′ : 5′-TCATTGTCCCAGCTGCGTGG-3′ (SEQ ID NO: 20)

The oligonucleotide primers were used for PCR amplification of twosegments of DNA of approximately 100 nts (primer pair 18) and 85 nts(primer pair 35) using 300 ngs of human genomic DNA as a template. ThePCR amplification was carried out as recommended by the manufacturer(Perkin-Elmer) under the following conditions: 35 cycles at 94° C. for60 sec, 55° C. for 50 sec and 72° C. for 30 sec. The PCR fragmentsobtained were purified from an agarose gel and re-amplified for 30cycles using the same PCR conditions described above.

After amplification, the PCR reactions were electrophoresed in agarosegels, the agarose containing the DNA bands of 100 and 85 ntsrespectively was excised, purified by QiaEx II (Qiagen), and then clonedinto plasmid pCR-script using Stratagene's pCR-Script cloning kit,followed by bacterial transformation and plating onto agar-ampicillinplates as recommended by the manufacturer. Bacterial colonies containingthe 100 and 85 nt inserts were identified by PCR using the primersdescribed above. The PCR fragments obtained were radiolabeled for use asprobes using a standard PCR reaction (Perkin-Elmer) on 20 ng of DNAtemplate, except that 5 nmoles each of dATP, dGTP and dTTP and 0.2mCurie of alpha ³²P-dCTP (Du Pont 3000 Ci/mmol) were added to thereaction mixture and then subjected to 7 cycles of PCR. Unincorporatedlabel was separated from the probes on a G50 NICK column (Pharmacia).These probes were used to screen a human fetal brain cDNA library(Stratagene Cat#936206) using standard library screening procedures(Sambrook, Fritsch and Maniatis, Molecular Cloning, a Laboratory Manual,(1989) Second Edition, Cold Spring Harbor Laboratory Press). One and ahalf million phage plaques were plated in XL-1 Blue bacteria asrecommended by Stratagene, and transferred to nitrocellulose filters induplicate as previously described (Id.). The filters were processed andeach filter replica was used for hybridization with one of the aboveprobes as previously described (Id.). Plaques hybridizing to both probeswere isolated and purified and a plasmid containing the cDNA insert wasexcised from the lambda clone according to Stratagene's recommendedprocedure (EXASSIST/SOLR System). The pBluescript plasmid containing thehuman Agrin insert was purified and the insert was then sequenced usingan automated sequencing kit (Applied Biosystems).

As a result of this screen, one clone (pBL-hAgrin1) was obtained whichcontains a nucleotide sequence encoding an amino acid sequence of humanagrin. The first amino acid encoded by the cloned nucleotide sequencecorresponds approximately to amino acid 424 of rat agrin (See FIGS.14A-14C). The nucleotide sequence of the clone ends downstream of thestop codon. Clone pBL-hAgrin1 contains a 4 amino acid insert starting atthe position which corresponds to position 1643 of FIGS. 14A-14C, apoint which was previously described for the rat as position “Y” (Stone,D. M. and Nikolics, K., J. Neurosci. 15: 6767-6778 (1995)). The sequenceof the 4 amino acid insert both in clone pBL-hAgrin1 and in the rat isKSRK.

A second clone was obtained from this screen. This second clone(pBL-hAgrin23) also contains a nucleotide sequence encoding an aminoacid sequence of human agrin. The first amino acid encoded by the clonednucleotide sequence corresponds approximately to amino acid 1552 of therat agrin (See FIGS. 14A-14C). (SEQ ID NO:34). The nucleotide sequenceof the clone ends downstream of the stop codon. Clone pBL-hAgrin23contains an 8 amino acid insert starting at a position which correspondsto position 1780 of FIGS. 14A-14C (SEQ ID NO: 34), a point which waspreviously described for the rat as position “Z” (Stone, D. M. andNikolics, K., J. Neurosci. 15: 6767-6778 (1995)). The sequence of theeight amino acid insert both in clone pBL-hAgrin23 and in the rat isELANEIPV. As previously discussed, it has been reported that the 8 aminoacid insert plays an important role in regulating the AChR clusteringactivity of different agrin forms. Therefore, by inserting a nucleotidesequence encoding the eight amino acid sequence ELANEIPV into clonepBL-hAgrin1 at the position corresponding to position Z of rat agrin, ahuman 4-8 agrin clone may be obtained. The addition of the 8 amino acidinsert at position Z should confer a high level of biological activityto the human 4-8 clone.

Clone pBL-hAgrin23 also contains the 4 amino acid “Y” insert asdescribed above for clone pBL-hAgrin1. However, clone pBL-hAgrin23contains 17 extra amino acids at the same “Y” position, such that thesequence of the “Y” insert in clone pBL-hAgrin23 isKSRKVLSASHPLTVSGASTPR. Therefore, in addition to the (4-0) and (4-8)human agrin splice variants described above, human clones correspondingto splice variants containing (Y-Z) inserts of (17-0), (17-8), (21-0),and (21-8) are indicated by these results and are within the scope ofthe present invention.

EXAMPLE 15 Expression of Human Agrin

Construction of Human Agrin Expression Vector

A human agrin Sfi I—Aat II fragment containing the 4 amino acid insertat the position corresponding to the Y-site described for rat agrin (seeFIGS. 14A-14C) was excised from clone pBL h agrin-1. A human agrin AatII—Not I fragment containing the 8 amino acid insert at the positioncorresponding to the Z-site described for rat agrin (see FIGS. 14A-14C)was excised from clone pBL h agrin-23. A Xho I—Sfi I fragment was thengenerated via PCR that contained a preprotrypsin signal peptide, the 8amino acid flg peptide (from the flag tagging system, IBI/Kodak,Rochester, N.Y.) and the human agrin sequence corresponding to thesequence of amino acids from position 1480 to the Sfi I site located atamino acids 1563-1566 of rat agrin (see FIGS. 14A-14C). The threefragments were then ligated into a Xho I—Not I digested pMT21 expressionvector to form the human agrin 4-8 expression vector pMT21-agrin 4-8.The sequence of human agrin 4-8 that was encoded in the expressionvector is shown in FIGS. 15A-15B (SEQ ID NO: 35). Expression vectors forthe human corresponding to splice variants containing (Y-Z) inserts of(0-8) and (4-0) were also constructed.

Expression of Human Agrin (4-8) in E. coli

The gene for human agrin 4-8 was PCR amplified from pMT21-agrin 4-8 withthe primer pair AG5′ (5′-GAGAGAGGTTTAAACATGAGCCCCTGCCAGCCCAACCCCTG-3′)and AG3′ (5′-CTCTGCGGCCGCTTATCATGGGGTGGGGCAGGGCCGCAG-3′) The PCR productwas digested with the restriction enzymes Pme I and Not I and clonedinto the Pme I and Not I sites of the vector pRG501, a pMB1 repliconthat confers kanamycin resistance and is designed to express clonedgenes from the phage T7 promoter. One isolate was characterized andnamed pRG531. The 1315 base pair Nco I-Nae I fragment internal to agrinin pRG531 was then replaced with the corresponding fragment frompMT21-agrin 4-8. The resulting plasmid, pRG451, was transformed into theexpression strain RFJ209 [IN(rrnD-rrn/E)1 lacI^(Q) lacZpL8 fhuAD322-405rpoS_((MC4100)) ara::(lacUV5-T7 gene 1)8]. Cultures of RFJ209/pRG541induced with IPTG express human agrin to about 5% of total cellularprotein and fractionates with soluble protein upon celdisruption. Thecrude soluble protein fraction containing human agrin 4-8, as well ashuman agrin 4-8 purified by Q-Sepharose chromatography was determined tobe active in phosphorylation of MuSK receptor.

Expression of Human Agrin (4-8) in Pichia pastoris

The 50 kD active fragment (portion) of human agrin 4-8 was cloned by PCRusing a primer containing a portion of the S. cerevisiae α mating factorpre-pro secretion signal and the 5′ end of the region encoding the 50 kDagrin fragment (GTATCTCTCGAGAAAAGAGAGGCTGAAGCT AGCCCCTGCCAGCCCAACC), anda primer containing sequences from the region 3′ of the agrin codingregion and a NotI site (AATAGTGCGGCCGCCAACACTCAGGCAAGAAAATCATATC). AfterPCR the fragment was digested with XhoI, which recognizes sequences inthe 5′ primer, and NotI, and was cloned into pPIC9 (Invitrogen) digestedwith XhoI and NotI. The resulting clone was digested with NotI andpartially digested with NcoI to remove most of the PCRed agrinsequences. This region was replaced by a NotI-NcoI fragment of agrinfrom pRG541. PCRed regions were sequenced and shown to be wild-type.This clone, pRG543 was digested with SalI and transformed into Pichiapastoris by electroporation. Transformants were selected for a His+ Mut+phenotype. Induction of the AOX1 promoter driving the expression ofhAgrin was done by growing the cells in buffered glycerol-complex mediumcontaining 0.5% glycerol, pH=6.0, for 24 hrs until the glycerol wasexhausted, at which point methanol was added to a final concentration of0.5%. The culture was centrifuged and the supernatant was dialyzedagainst PBS. The concentration of hAgrin was approximately 10 ug/ml andwas determined to be active in phosphorylation of MuSK receptor.

Production of Human Agrin 4,8 from Baculovirus Infected Insect Cells

Virus Production

The flg-tagged gene for human agrin 4-8 was engineered into abaculovirus expression plasmid and recombined with viral DNA to generaterecombinant baculovirus, amplified and harvested using methodspreviously described (O'Reilly, D. R., L. K. Miller, and V. A. Luckow,Baculovirus Expression Vectors—A Laboratory Manual 1992, New York: W. H.Freeman). SF21 insect cells (Spodoptera frugiperda) obtained fromInvitrogen were adapted and expanded at 27° C. in Gibco SF900 IIserum-free medium. Uninfected cells were grown to a density of 1×10⁶cells/mL. Cell density was determined by counting viable cells using ahemacytometer. The virus stock for FLAG-agrin was added to thebioreactor at a low multiplicity 0.01-0.1 PFU/cell to begin theinfection. The infection process was allowed to continue for 3-4 daysallowing maximum virus replication without incurring substantial celllysis. The cell suspension was aseptically aliquoted into sterilecentrifuge bottles and the cells removed by centrifugation (1600 RPM, 30min). The cell-free supernatant was collected in sterile bottles andstored at 4° C. in the absence of light until further use.

The virus titer was determined by plaque assay as described by O'Reilly,Miller and Luckow. The method is carried out in 60 mm tissue-culturedishes which are seeded with 1.5×10⁶ cells. Serial dilutions of thevirus stock are added to the attached cells and the mixture incubatedwith rocking to allow the virus to adsorb to individual cells. An agaroverlay is added and plates incubated for 5 days at 27° C. Viable cellswere stained with neutral red revealing circular plaques which werecounted to give the virus titer expressed in plaque forming unit permilliliter (PFU/mL).

Infection of Cells for Protein Production

Uninfected SF21 cells were grown in a 60 L ABEC bioreactor containing 40L of Gibco SF900 II medium with gentamicin sulfate (25 mg/L) andamphotericin B (1 mg/L). Temperature was controlled at 27 C and thedissolved oxygen level was maintained at 50% of saturation bycontrolling the flowrate of oxygen in the inlet gas stream. When adensity of 2×10⁶ cells/mL was reached, the cells were concentratedwithin the bioreactor to 20 L, using a low shear steam sterilizable pumpand a tangential flow filtration device with Millipore Prostak 0.45micron membranes. After concentration, fresh sterile growth medium wasslowly added to the bioreactor while the filtration system continued toremove the spent growth medium by diafiltration. After two volumeexchanges an additional 20 L of fresh medium was added to the bioreactorto resuspend the cells to the original volume of 40 L.

The amount of virus stock required was calculated based on the celldensity, virus titer and the desired multiplicity of infection (MOI).Multiplicity ratios between 1 and 10 pfu/cell have been effectivelyused. The virus stock was added aseptically to the bioreactor and theinfection was allowed to proceed for three to four days.

Recovery and Chromatographic Purification

At the conclusion of the infection phase of the bioreactor process thecells were concentrated in the bioreactor using a 30 ft² MilliporeProstak filter (0.45 micron) pore size. The cell-free permeate passingthrough the filter was collected in a clean process vessel. The proteinwas diafiltered into a low conductivity buffer (20 mM citrate, pH 5.5)using Millipore Pellicon ultrafiltration membrane cassettes totaling 20ft² with a nominal 10 kiloDalton cutoff. The protein in the retentatewas loaded onto a cation exchange column (Pharmacia SP Sepharose FF)equilibrated with 20 mM citrate buffer, pH 5.5. After loading theprotein was washed first with 20 mM citrate, 200 mM sodium chloride, pH5.5 then with 20 mM Bicine, pH 8.0 to remove contaminating proteins. Theprotein was eluted with a 0-750 mM sodium chloride linear gradient over7.5 column volumes. The eluted agrin was buffer exchanged into 20 mMTris, pH 8.5 buffer to remove salt for subsequent binding to an anionexchange column.

The agrin was then bound to a Pharmacia Q Sepharose FF columnequilibrated with 20 mM Tris, pH 8.5. After loading the column waswashed with the same buffer to remove contaminating proteins and theprotein eluted with a 0-250 mM sodium chloride gradient. The fractionscontaining agrin were pooled and concentrated and dialyzed into PBScontaining calcium and magnesium.

Expression of Human Agrin (4-8) in COS-7 Cells

Lipofectamine reagent (GIBCO-BRL, Inc.) and recommended protocols wereused to transfect COS-7 cells with the human agrin cDNA clonepMT21-agrin 4-8 containing a nucleotide sequence encoding the eightamino acid sequence ELANEIPV at the position corresponding to position Zof rat agrin. COS media containing secreted ligand was harvested afterthree days and concentrated 20-fold by diafiltration (DIAFLOultrafiltration membranes, Amicon, Inc.). The quantity of active humanagrin present in the media was determined and expressed as the amount(in resonance units, R.U.) of MuSK receptor specific binding activitymeasured by a BIAcore binding assay.

EXAMPLE 16 Preparation of Truncated Molecules Containing the MuSKActivation Portion of Human Agrin

It has recently been reported that a 21 kD fragment of chick agrin issufficient to induce AChR aggregation (Gesemann, M., et al., 1995, J.Cell. Biol. 128: 625-636). Applicants therefore decided to investigatethe properties of various portions of human agrin and to test theability of each to induce phosphorylation of the MuSK receptor.

As set forth in FIGS. 15A-15B (SEQ ID NO: 36), the amino acid sequenceof the 50 kD active portion of human agrin 4,8 is 492 amino acids long.A preprotrypsin signal sequence (Stevenson et al., 1986. Nucleic AcidsRes. 21: 8307-8330) precedes a FLAG tag sequence (Hopp et al. 1988.Bio/Technology 6: 1204-1210); together, they constitute the first 23amino acids. Thus the agrin 4,8 sequence begins with amino acid 24.Truncated molecules were created, each of which contained the signalsequence and FLAG tag (23 amino acids) followed by the agrin 4,8sequence to which N-terminal deletions had been made to create portionsof agrin (designated delta 3 through 9) as follows:

-   delta 3: agrin sequence starts with amino acid #60: QTAS . . . (SEQ    ID NO: 25)-   delta 4: agrin sequence starts with amino acid #76: NGFS . . . (SEQ    ID NO: 26)-   delta 5: agrin sequence starts with amino acid #126: VSLA . . . (SEQ    ID NO: 27)-   delta 6: agrin sequence starts with amino acid #178: GPRV . . . (SEQ    ID NO: 28)-   delta 7: agrin sequence starts with amino acid #222: GFDG . . . (SBQ    ID NO: 29)-   delta 8: agrin sequence starts with amino acid #260: ASGH . . . (SEQ    ID NO: 30)-   delta 9: agrin sequence starts with amino acid #300: AGDV . . . (SEQ    ID NO: 31)

All of the sequences continue to the terminal amino acids PCPTP, as withthe 50 kD agrin.

The truncated molecules were made as follows: PCR primers were designedconsistent with the nucleotide sequences encoding the first and last tenamino acids of each construct. Included in the 5′ primer was sequencedata to append the preprotrypsin signal sequence and “FLAG-tag” to theamino terminus of each agrin fragment. Thus, the shortest truncatedmolecule (delta 9) contains the signal sequence and FLAG tag and thehuman agrin sequence from amino acid 300 to 492 of human c-agrin 4,8.DNA encoding the “delta” forms of truncated c-agrin 4,8 was then clonedinto a eukaryotic expression vector, and transient transfections wereperformed as previously described (Glass, D., et al., 1991, Cell 66:405-413; Ip, N.Y., et al., 1992, PNAS (USA) 89: 3060-3064).

Agrin levels in the COS-transfected supernatants were determined byWestern analysis of the conditioned media with an agrin-specificantibody (Stressgen 131), using a purified agrin control of knownconcentration. Each of the truncated molecules was expressed and shownto be capable of inducing tyrosine phosphorylation of MuSK receptor.

FIG. 16 shows that the delta 9 truncated molecule can inducephosphorylation of the MuSK receptor, though with less efficiency thanthe 50 kD agrin 4,8 molecule. Thus, each of the truncated moleculescreated exhibited the biological activity of human agrin 4,8 withrespect to the MuSK receptor.

EXAMPLE 17 PEGylation of Agrin 4,8 and Pharmacokinetics Study

After intravenous administration, the 50 kD agrin 4,8 was clearedrapidly from the systemic circulation with a half-life of <10 minutes.(See FIG. 17). It was known that the properties of certain proteins canbe modulated by attachment of polyethylene glycol (PEG) polymers, whichincreases the hydrodynamic volume of the protein and thereby slows itsclearance by kidney filtration. (See, e.g. Clark, R., et al., 1996, J.Biol. Chem. 271: 21969-21977). Therefore, in an attempt to increase itscirculating half-life, agrin 4,8 was modified by covalent attachment ofa polyethylene glycol molecule and the effect on the protein's serumhalf-life was studied.

A solution of 500 mg of human agrin 4,8 at 3.0 mg/mL was added to areaction vessel and the PEGylation reaction was initiated by addition ofmonomethoxypoly-ethyleneglycol succinimidyl propionate (PEG)(approximate molecular weight=20,000 daltons). A ratio of 1.75 moles PEGreagent to agrin was used for the reaction which was carried out at a pHof 7.3 to 8.5 in phosphate buffered saline over a period of 2 hours. Thereaction was stopped by addition of 50 mM Tris hydrochloride.

The PEGylated agrin was diluted with buffer at a pH of 8.2 to lower boththe conductivity and the concentration of Tris, before loading onto acation exchange column (Pharmacia SP High Performance). The column waseluted using a gradient from 0 to 600 mM sodium chloride in Tris bufferat a pH of 8.2. Numerous distinct forms of PEGylated agrin eluted alongthe gradient and unmodified agrin eluted at the high salt end of thegradient. Monopegylated forms were selectively pooled for subsequent invivo testing.

Adult Sprague-Dawley rats (male or female) weighing 300-500 g wereanesthetized with ketamine/xylazine (50/10 mg/kg) and the right hindlimb muscles were denervated by transecting the sciatic nerve at midthigh level. After 10-14 days (when MuSK expression was substantiallyelevated in denervated muscle) rats were re-anesthetized and the rightjugular vein was exposed by cut down surgery. Rats were thenadministered doses of agrin 4,8 or PEG-agrin 4,8 ranging from 1-10 mg/kginto the jugular vein with a 27 gauge needle. After injection, the woundwas sutured and tail blood samples were taken at 0 (pre-injection), 5,10, 15, 30 minutes, and at 1, 2, 4, 6, 8, 16, 24, and 48 hours after theinjection. Serum samples were harvested from centrifuged blood andassayed using an ELISA. Serum levels are expressed as μg/ml of serum.

As shown in FIG. 17, at a dose of 10 mg/kg, i.v., agrin 4,8 was rapidlycleared from the blood with a half-life of ˜10 minutes. In contrast, thehalf-life of PEG-agrin 4,8 (also at 10 mg/kg, i.v.) was dramaticallyincreased by ˜10-fold. These results show that modification of agrin 4,8with PEG greatly increases its apparent half-life in the blood. Thus,PEGylated agrin may have prolonged activity on MuSK in denervatedskeletal muscle and may thus be a more effective treatment for musculardisorders and conditions such as muscle atrophy. It is expected that thePEGylation of the above-described truncated agrin molecules wouldsimilarly increase their apparent half-lives in the blood.

Deposit of Microorganisms

A clone designated pBluescript SK-containing dmk was deposited with theAmerican Type Culture Collection, 12301 Parklawn Drive, Rockville, Md.20852 on Jul. 13, 1993 under ATCC Accession No. 75498. RecombinantAutographa californica baculovirus encoding the rat Dmk RB (i.e., ratMuSK-IgG1 receptorbody) was designated “vDmk receptorbody” and depositedwith the American Type Culture Collection, 12301 Parklawn Drive,Rockville, Md. 20852 on May 16, 1995 under ATCC Accession No. VR-2507.The cDNA clone pBL-hAgrin1 was deposited with the American Type CultureCollection, 12301 Parklawn Drive, Rockville, Md. 20852 on Dec. 12, 1995under ATCC Accession No. 97378.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingfigures. Such modifications are intended to fall within the scope of theappended claims.

1. A human agrin protein capable of inducing phosphorylation of the MuSKreceptor, and fragments of human agrin which retain the capacity ofinducing phosphorylation of the MuSK receptor, selected from the groupconsisting of SEQ ID NOs:25-31 and
 36. 2. The protein of claim 1 whichis pegylated.
 3. A composition comprising the protein of claim 1 and anacceptable carrier.